Small volume in vitro analyte sensor and methods of making

ABSTRACT

A sensor utilizing a non-leachable or diffusible redox mediator is described. The sensor includes a sample chamber to hold a sample in electrolytic contact with a working electrode, and in at least some instances, the sensor also contains a non-leachable or a diffusible second electron transfer agent. The sensor and/or the methods used produce a sensor signal in response to the analyte that can be distinguished from a background signal caused by the mediator. The invention can be used to determine the concentration of a biomolecule, such as glucose or lactate, in a biological fluid, such as blood or serum, using techniques such as coulometry, amperometry; and potentiometry. An enzyme capable of catalyzing the electrooxidation or electroreduction of the biomolecule is typically provided as a second electron transfer agent.

This application is a continuation of Ser. No. 11/734,979, filed on Apr.13, 2007, which is a divisional of U.S. application Ser. No. 10/662,081,filed Sep. 12, 2003, now issued as U.S. Pat. No. 7,225,535, which is adivisional application of U.S. application Ser. No. 09/594,285, filedJun. 15, 2000, now issued as U.S. Pat. No. 6,618,934, which is acontinuation of U.S. application Ser. No. 09/295,962, filed Apr. 21,1999, now issued as U.S. Pat. No. 6,338,790, which claims the benefit ofU.S. Provisional Application Ser. No. 60/103,627, filed Oct. 8, 1998 andU.S. Provisional Application Ser. No. 60/105,773, filed Oct. 8, 1998.

FIELD OF THE INVENTION

This invention relates to analytical sensors for the detection ofbioanalytes in a small volume sample.

BACKGROUND OF THE INVENTION

Analytical sensors are useful in chemistry and medicine to determine thepresence and concentration of a biological analyte. Such sensors areneeded, for example, to monitor glucose in diabetic patients and lactateduring critical care events.

Currently available technology measures bioanalytes in relatively largesample volumes, e.g., generally requiring 3 microliters or more of bloodor other biological fluid. These fluid samples are obtained from apatient, for example, using a needle and syringe, or by lancing aportion of the skin such as the fingertip and “milking” the area toobtain a useful sample volume. These procedures are inconvenient for thepatient, and often painful, particularly when frequent samples arerequired. Less painful methods for obtaining a sample are known such aslancing the arm or thigh, which have a lower nerve ending density.However, lancing the body in the preferred regions typically producessubmicroliter samples of blood, because these regions are not heavilysupplied with near-surface capillary vessels.

It would therefore be desirable and very useful to develop a relativelypainless, easy to use blood analyte sensor, capable of performing anaccurate and sensitive analysis of the concentration of analytes in asmall volume of sample.

Sensors capable of electrochemically measuring an analyte in a sampleare known in the art. Some sensors known in the art use at least twoelectrodes and may contain a redox mediator to aid in theelectrochemical reaction. However, the use of an electrochemical sensorfor measuring analyte in a small volume introduces error into themeasurements. One type of inaccuracy arises from the use of a diffusibleredox mediator. Because the electrodes are so close together in a smallvolume sensor, diffusible redox mediator may shuttle between the workingand counter electrode and add to the signal measured for analyte.Another source of inaccuracy in a small volume sensor is the difficultyin determining the volume of the small sample or in determining whetherthe sample chamber is filled. It would therefore be desirable to developa small volume electrochemical sensor capable of decreasing the errorsthat arise from the size of the sensor and the sample.

SUMMARY OF THE INVENTION

The sensors of the present invention provide a method for the detectionand quantification of an analyte in submicroliter samples. In general,the invention includes a method and sensor for analysis of an analyte ina small volume of sample by, for example, coulometry, amperometry and/orpotentiometry. A sensor of the invention utilizes a non-leachable ordiffusible redox mediator. The sensor also includes a sample chamber tohold the sample in electrolytic contact with the working electrode. Inmany instances, the sensor also contains a non-leachable or diffusiblesecond electron transfer agent.

In a preferred embodiment, the working electrode faces a counterelectrode, forming a measurement zone within the sample chamber, betweenthe two electrodes, that is sized to contain no more than about 1 μL ofsample, preferably no more than about 0.5, more preferably no more thanabout 0.25 μL, and most preferably no more than about 0.1 μL of sample.A sorbent material is optionally positioned in the sample chamber andmeasurement zone to reduce the volume of sample needed to fill thesample chamber and measurement zone.

In one embodiment of the invention, a biosensor is provided whichcombines coulometric electrochemical sensing with a non-leachable ordiffusible redox mediator to accurately and efficiently measure abioanalyte in a submicroliter volume of sample. The preferred sensorincludes an electrode, a non-leachable or diffusible redox mediator onthe electrode, a sample chamber for holding the sample in electricalcontact with the electrode and, preferably, sorbent material disposedwithin the sample chamber to reduce the volume of the chamber. Thesample chamber, together with any sorbent material, is sized to providefor analysis of a sample volume that is typically no more than about 1μL, preferably no more than about 0.5 μL, more preferably no more thanabout 0.25 μL, and most preferably no more than about 0.1 μL. In someinstances, the sensor also contains a non-leachable or diffusible secondelectron transfer agent.

One embodiment of the invention includes a method for determining theconcentration of an analyte in a sample by, first, contacting the samplewith an electrochemical sensor and then determining the concentration ofthe analyte. The electrochemical sensor includes a facing electrode pairwith a working electrode and a counter electrode and a sample chamber,including a measurement zone, positioned between the two electrodes. Themeasurement zone is sized to contain no more than about 1 μL of sample.

The invention also includes an electrochemical sensor with two or morefacing electrode pairs. Each electrode pair has a working electrode, acounter electrode, and a measurement zone between the two electrodes,the measurement zone being sized to hold no more than about 1 μL ofsample. In addition, the sensor also includes a non-leachable redoxmediator on the working electrode of at least one of the electrode pairsor a diffusible redox mediator on a surface in the sample chamber or inthe sample.

One aspect of the invention is a method of determining the concentrationof an analyte in a sample by contacting the sample with anelectrochemical sensor and determining the concentration of the analyteby coulometry. The electrochemical sensor includes an electrode pairwith a working electrode and a counter electrode. The sensor alsoincludes a sample chamber for holding a sample in electrolytic contactwith the working electrode. Within the sample chamber is sorbentmaterial to reduce the volume sample needed to fill the sample chamberso that the sample chamber is sized to contain no more than about 1 μLof sample. The sample chamber also contains a non-leachable ordiffusible redox mediator and optionally contains a non-leachable ordiffusible second electron transfer agent.

The sensors may also include a fill indicator, such as an indicatorelectrode or a second electrode pair, that can be used to determine whenthe measurement zone or sample chamber has been filled. An indicatorelectrode or a second electrode pair may also be used to increaseaccuracy of the measurement of analyte concentration. The sensors mayalso include a heating element to heat the measurement zone or samplechamber to increase the rate of oxidation or reduction of the analyte.

Sensors can be configured for side-filling or tip-filling. In addition,in some embodiments, the sensor may be part of an integrated sampleacquisition and analyte measurement device. The integrated sampleacquisition and analyte measurement device may include the sensor and askin piercing member, so that the device can be used to pierce the skinof a user to cause flow of a fluid sample, such as blood, that can thenbe collected by the sensor. In at least some embodiments, the fluidsample can be collected without moving the integrated sample acquisitionand analyte measurement device.

One method of forming a sensor, as described above, includes forming atleast one working electrode on a first substrate and forming at leastone counter or counter/reference electrode on a second substrate. Aspacer layer is disposed on either the first or second substrates. Thespacer layer defines a channel into which a sample can be drawn and heldwhen the sensor is completed. A redox mediator and/or second electrontransfer agent are disposed on the first or second substrate in a regionthat will be exposed within the channel when the sensor is completed.The first and second substrates are then brought together and spacedapart by the spacer layer with the channel providing access to the atleast one working electrode and the at least one counter orcounter/reference electrode. In some embodiments, the first and secondsubstrates are portions of a single sheet or continuous web of material.

These and various other features which characterize the invention arepointed out with particularity in the attached claims. For a betterunderstanding of the invention, its advantages, and objectives obtainedby its use, reference should be made to the drawings and to theaccompanying description, in which there is illustrated and describedpreferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings, wherein like reference numerals andletters indicate corresponding structure throughout the several views:

FIG. 1 is a schematic view of a first embodiment of an electrochemicalsensor in accordance with the principles of the present invention havinga working electrode and a counter electrode facing each other;

FIG. 2 is a schematic view of a second embodiment of an electrochemicalsensor in accordance with the principles of the present invention havinga working electrode and a counter electrode in a coplanar configuration;

FIG. 3 is a schematic view of a third embodiment of an electrochemicalsensor in accordance with the principles of the present invention havinga working electrode and a counter electrode facing each other and havingan extended sample chamber;

FIG. 4 is a not-to-scale side-sectional drawing of a portion of thesensor of FIG. 1 or 3 showing the relative positions of the redoxmediator, the sample chamber, and the electrodes;

FIG. 5 is a top view of a fourth embodiment of an electrochemical sensorin accordance with the principles of the present invention, this sensorincludes multiple working electrodes;

FIG. 6 is a perspective view of an embodiment of an analyte measurementdevice, in accordance with the principles of the present invention,having a sample acquisition means and the sensor of FIG. 4;

FIG. 7 is a graph of the charge required to electrooxidize a knownquantity of glucose in an electrolyte buffered solution (filled circles)or serum solution (open circles) using the sensor of FIG. 1 with glucoseoxidase as the second electron transfer agent;

FIG. 8 is a graph of the average glucose concentrations for the data ofFIG. 7 (buffered solutions only) with calibration curves calculated tofit the averages; a linear calibration curve was calculated for the10-20 mM concentrations and a second order polynomial calibration curvewas calculated for the 0-10 mM concentrations;

FIG. 9 is a Clarke-type clinical grid analyzing the clinical relevanceof the glucose measurements of FIG. 7;

FIG. 10 is a graph of the charge required to electrooxidize a knownquantity of glucose in an electrolyte buffered solution using the sensorof FIG. 1 with glucose dehydrogenase as the second electron transferagent;

FIGS. 11A, 11B, and 11C are top views of three configurations foroverlapping working and counter electrodes according to the presentinvention;

FIGS. 12A and 1213 are cross-sectional views of one embodiment of anelectrode pair formed using a recess of a base material, according tothe invention;

FIGS. 13A and 13B are cross-sectional views of yet another embodiment ofan electrode pair of the present invention formed in a recess of a basematerial;

FIGS. 14A and 14B are cross-sectional views of a further embodiment ofan electrode pair of the present invention formed using a recess of abase material and a sorbent material;

FIG. 15 is a graph of charge delivered by a sensor having a diffusibleredox mediator over time for several concentrations of glucose;

FIG. 16 is a graph of charge delivered by a sensor having a diffusibleredox mediator for several glucose concentrations;

FIG. 17 is a graph of charge delivered by sensors with different amountsof diffusible redox mediator over time.

FIG. 18A illustrates a top view of a′ first film with a workingelectrode for use in a fifth embodiment of a sensor according to theinvention;

FIG. 18B illustrates a top view of a spacer for placement on the firstfilm of FIG. 18A;

FIG. 18C illustrates a bottom view of a second film (inverted withrespect to FIGS. 18A and 18B) with counter electrodes placement over thespacer of FIG. 18B and first film of FIG. 18A;

FIG. 19A illustrates a top view of a first film with a working electrodefor use in a sixth embodiment of a sensor according to the invention;

FIG. 19B illustrates a top view of a spacer for placement on the firstfilm of FIG. 19A;

FIG. 19C illustrates a bottom view of a second film (inverted withrespect to FIGS. 19A and 1913) with counter electrodes placement overthe spacer of FIG. 19B and first film of FIG. 19A;

FIG. 20A illustrates a top view of a first film with a working electrodefor use in a seventh embodiment of a sensor according to the invention;

FIG. 20B illustrates a top view of a spacer for placement on the firstfilm of FIG. 20A;

FIG. 20C illustrates a bottom view of a second film (inverted withrespect to FIGS. 20A and 20B) with counter electrodes placement over thespacer of FIG. 20B and first film of FIG. 20A;

FIG. 21A illustrates a top view of a first film with a working electrodefor use in a eighth embodiment of a sensor according to the invention;

FIG. 21B illustrates a top view of a spacer for placement on the firstfilm of FIG. 21A;

FIG. 21C illustrates a bottom view of a second film (inverted withrespect to FIGS. 21A and 21B) with counter electrodes placement over thespacer of FIG. 21B and first film of FIG. 21A;

FIG. 22A illustrates a top view of a first film with a working electrodefor use in a ninth embodiment of a sensor according to the invention;

FIG. 22B illustrates a top view of a spacer for placement on the firstfilm of FIG. 22A;

FIG. 22C illustrates a bottom view of a second film (inverted withrespect to FIGS. 22A and 22B) with counter electrodes placement over thespacer of FIG. 22B and first film of FIG. 22A;

FIG. 23A illustrates a top view of a first film with a working electrodefor use in a tenth embodiment of a sensor according to the invention;

FIG. 23B illustrates a top view of a spacer for placement on the firstfilm of FIG. 23A;

FIG. 23C illustrates a bottom view of a second film (inverted withrespect to FIGS. 23A and 23B) with counter electrodes placement over thespacer of FIG. 23B and first film of FIG. 23A;

FIG. 24A illustrates a top view of a first film with a working electrodefor use in an eleventh embodiment of a sensor according to theinvention;

FIG. 24B illustrates a top view of a spacer for placement on the firstfilm of FIG. 24A;

FIG. 24C illustrates a bottom view of a second film (inverted withrespect to FIGS. 24A and 24B) with counter electrodes placement over thespacer of FIG. 24B and first film of FIG. 24A;

FIG. 25 illustrates a top view of a twelfth embodiment of anelectrochemical sensor, according to the invention;

FIG. 26 illustrates a perspective view of one embodiment of anintegrated analyte acquisition and sensor device;

FIG. 27 illustrates a cross-sectional view of a thirteenth embodiment ofa sensor, according to the invention;

FIG. 28 illustrates a graph comparing measurements of analyteconcentration in blood samples collected from a subject's arm made by asensor of the invention with those determined by a standard blood test;

FIG. 29 illustrates a graph comparing measurements of analyteconcentration in blood samples collected from a subject's finger made bya sensor of the invention with those determined by a standard bloodtest;

FIG. 30 illustrates a graph comparing measurements of analyteconcentration in venous samples made by a sensor of the invention withthose determined by a standard blood test;

FIG. 31A illustrates a top view of one embodiment of a sheet of sensorcomponents, according to the invention;

FIG. 31B illustrates a top view of another embodiment of a sheet ofsensor components, according to the invention; and

FIG. 32 illustrates a cross-sectional view looking from inside the meterto a sensor of the invention disposed in a meter.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

When used herein, the following definitions define the stated term:

An “air-oxidizable mediator” is a redox mediator that is oxidized byair, preferably so that at least 90% of the mediator is in an oxidizedstate upon storage in air either as a solid or as a liquid during aperiod of time, for example, one month or less, and, preferably, oneweek or less, and, more preferably, one day or less.

“Amperometry” includes steady-state amperometry, chronoamperometry, andCottrell-type measurements.

A “biological fluid” is any body fluid in which the analyte can bemeasured, for example, blood, interstitial fluid, dermal fluid, sweat,and tears.

The term “blood” in the context of the invention includes whole bloodand its cell-free components, such as, plasma and serum.

“Coulometry” is the determination of charge passed or projected to passduring complete or nearly complete electrolysis of the analyte, eitherdirectly on the electrode or through one or more electron transferagents. The charge is determined by measurement of charge passed duringpartial or nearly complete electrolysis of the analyte or, more often,by multiple measurements during the electrolysis of a decaying currentand elapsed time. The decaying current results from the decline in theconcentration of the electrolyzed species caused by the electrolysis.

A “counter electrode” refers to one or more electrodes paired with theworking electrode, through which passes an electrochemical current equalin magnitude and opposite in sign to the current passed through theworking electrode. The term “counter electrode” is meant to includecounter electrodes which also function as reference electrodes (i.e. acounter/reference electrode) unless the description provides that a“counter electrode” excludes a reference or counter/reference electrode.

An “effective diffusion coefficient” is the diffusion coefficientcharacterizing transport of a substance, for example, an analyte, anenzyme, or a redox mediator, in the volume between the electrodes of theelectrochemical cell. In at least some instances, the cell volume may beoccupied by more than one medium (e.g., the sample fluid and a polymerfilm). Diffusion of a substance through each medium may occur at adifferent rate. The effective diffusion coefficient corresponds to adiffusion rate through this multiple-media volume and is typicallydifferent than the diffusion coefficient for the substance in a cellfilled solely with sample fluid.

An “electrochemical sensor” is a device configured to detect thepresence of and/or measure the concentration of an analyte viaelectrochemical oxidation and reduction reactions. These reactions aretransduced to an electrical signal that can be correlated to an amountor concentration of analyte.

“Electrolysis” is the electrooxidation or electroreduction of a compoundeither directly at an electrode or via one or more electron transferagents (e.g., redox mediators and/or enzymes).

The term “facing electrodes” refers to a configuration of the workingand counter electrodes in which the working surface of the workingelectrode is disposed in approximate opposition to a surface of thecounter electrode. In at least some instances, the distance between theworking and counter electrodes is less than the width of the workingsurface of the working electrode.

A compound is “immobilized” on a surface when it is entrapped on orchemically bound to the surface.

An “indicator electrode” includes one or more electrodes that detectpartial or complete filling of a sample chamber and/or measurement zone.

A “layer” includes one or more layers.

The “measurement zone” is defined herein as a region of the samplechamber sized to contain only that portion of the sample that is to beinterrogated during an analyte assay.

A “non-diffusible,” “non-leachable,” or “non-releasable” compound is acompound which does not substantially diffuse away from the workingsurface of the working electrode for the duration of the analyte assay.

The “potential of the counter/reference electrode” is the half cellpotential of the reference electrode or counter/reference electrode ofthe cell when the solution in the cell is 0.1 M NaCl solution at pH7.

“Potentiometry” and “chronopotentiometry” refer to taking apotentiometric measurement at one or more points in time.

A “redox mediator” is an electron transfer agent for carrying electronsbetween the analyte and the working electrode, either directly, or via asecond electron transfer agent.

A “reference electrode” includes a reference electrode that alsofunctions as a counter electrode (i.e., a counter/reference electrode)unless the description provides that a “reference electrode” excludes acounter/reference electrode.

A “second electron transfer agent” is a molecule that carries electronsbetween a redox mediator and the analyte.

“Sorbent material” is material that wicks, retains, and/or is wetted bya fluid sample and which typically does not substantially preventdiffusion of the analyte to the electrode.

A “surface in the sample chamber” includes a surface of a workingelectrode, counter electrode, counter/reference electrode, referenceelectrode, indicator electrode, a spacer, or any other surface boundingthe sample chamber.

A “working electrode” is an electrode at which analyte iselectrooxidized or electroreduced with or without the agency of a redoxmediator.

A “working surface” is the portion of a working electrode that iscovered with non-leachable redox mediator and exposed to the sample, or,if the redox mediator is diffusible, a “working surface” is the portionof the working electrode that is exposed to the sample.

The small volume, in vitro analyte sensors of the present invention aredesigned to measure the concentration of an analyte in a portion of asample having a volume no more than about 1 μL, preferably no more thanabout 0.5 μL, more preferably no more than about 0.25 μL, and mostpreferably no more than about 0.1 μL. The analyte of interest istypically provided in a solution or biological fluid, such as blood orserum. Referring to the Drawings in general and FIGS. 1-4 in particular,a small volume, in vitro electrochemical sensor 20 of the inventiongenerally includes a working electrode 22, a counter (orcounter/reference) electrode 24, and a sample chamber 26 (see FIG. 4).The sample chamber 26 is configured so that when a sample is provided inthe chamber the sample is in electrolytic contact with both the workingelectrode 22 and the counter electrode 24. This allows electricalcurrent to flow between the electrodes to effect the electrolysis(electrooxidation or electroreduction) of the analyte.

Working Electrode

The working electrode 22 may be formed from a molded carbon fibercomposite or it may consist of an inert non-conducting base material,such as polyester, upon which a suitable conducting layer is deposited.The conducting layer typically has relatively low electrical resistanceand is typically electrochemically inert over the potential range of thesensor during operation. Suitable conducting layers include gold,carbon, platinum, ruthenium dioxide, palladium, and conductive epoxies,such as, for example, ECCOCOAT CT5079-3 Carbon-Filled Conductive EpoxyCoating (available from W.R. Grace Company, Woburn, Mass.), as well asother non-corroding materials known to those skilled in the art. Theelectrode (e.g., the conducting layer) is deposited on the surface ofthe inert material by methods such as vapor deposition or printing.

A tab 23 may be provided on the end of the working electrode 22 for easyconnection of the electrode to external electronics (not shown) such asa voltage source or current measuring equipment. Other known methods orstructures (such as contact pads) may be used to connect the workingelectrode 22 to the external electronics.

To prevent electrochemical reactions from occurring on portions of theworking electrode not coated by the mediator, when a non-leachablemediator is used, a dielectric 40 may be deposited on the electrodeover, under, or surrounding the region with the redox mediator, as shownin FIG. 4. Suitable dielectric materials include waxes andnon-conducting organic polymers such as polyethylene. Dielectric 40 mayalso cover a portion of the redox mediator on the electrode. The coveredportion of the redox mediator will not contact the sample, and,therefore, will not be a part of the electrode's working surface.

Sensing Chemistry

In addition to the working electrode 22, sensing chemistry materials areprovided in the sample chamber 26 for the analysis of the analyte. Thissensing chemistry preferably includes a redox mediator and a secondelectron transfer mediator, although in some instances, one or the othermay be used alone. The redox mediator and second electron transfer agentcan be independently diffusible or non-leachable (i.e., non-diffusible)such that either or both may be diffusible or non-leachable. Placementof sensor chemistry components may depend on whether they are diffusibleor non-leachable. For example, non-leachable and/or diffusiblecomponent(s) typically form a sensing layer on the working electrode.Alternatively, one or more diffusible components may be disposed on anysurface in the sample chamber prior to the introduction of the sample.As another example, one or more diffusible component(s) may be placed inthe sample prior to introduction of the sample into the sensor.

If the redox mediator is non-leachable, then the non-leachable redoxmediator is typically disposed on the working electrode 22 as a sensinglayer 32. In an embodiment having a redox mediator and a second electrontransfer agent, if the redox mediator and second electron transfer agentare both non-leachable, then both of the non-leachable components aredisposed on the working electrode 22 as a sensing layer 32.

If, for example, the second electron transfer agent is diffusible andthe redox mediator is non-leachable, then at least the redox mediator isdisposed on the working electrode 22 as a sensing layer 32. Thediffusible second electron transfer agent need not be disposed on asensing layer of the working electrode, but may be disposed on anysurface of the sample chamber, including within the redox mediatorsensing layer, or may be placed in the sample. If the redox mediator isdiffusible, then the redox mediator may be disposed on any surface ofthe sample chamber or may be placed in the sample.

If both the redox mediator and second electron transfer agent arediffusible, then the diffusible components may be independently orjointly disposed on any surface of the sample chamber and/or placed inthe sample (i.e., each diffusible component need not be disposed on thesame surface of the sample chamber or placed in the sample).

The redox mediator, whether it is diffusible or non-leachable, mediatesa current between the working electrode 22 and the analyte and enablesthe electrochemical analysis of molecules which may not be suited fordirect electrochemical reaction on an electrode. The mediator functionsas an electron transfer agent between the electrode and the analyte.

In one embodiment, the redox mediator and second electron transfer agentare diffusible and disposed on the same surface of the sample chamber,such as, for example, on the working electrode. In this same vein, bothcan be disposed on, for example, the counter electrode,counter/reference electrode, reference electrode, or indicatorelectrode. In other instances, the redox mediator and second electrontransfer agent are both diffusible and independently placed on a surfaceof the sample chamber and/or in the sample. For example, the redoxmediator may be placed on the working electrode while the secondelectron transfer agent is placed on any surface, except for the workingelectrode, or is placed in the sample. Similarly, the reverse situationin which the second electron transfer agent is disposed on the workingelectrode and the redox mediator is disposed on any surface, except forthe working electrode, or is placed in the sample is also a suitableembodiment. As another example, the redox mediator may be disposed onthe counter electrode and the second electron transfer agent is placedon any surface except for the counter electrode or is placed in thesample. The reverse situation is also suitable.

The diffusible redox mediator and/or second electron transfer agent maydiffuse rapidly into the sample or diffusion may occur over a period oftime. Similarly, the diffusible redox mediator and/or second electrontransfer agent may first dissolve from the surface on which it wasplaced as a solid and then the diffusible redox mediator and/or secondelectron transfer agent may diffuse into the sample, either rapidly orover a period of time. If the redox mediator and/or second electrontransfer agent diffuse over a period of time, a user may be directed towait a period of time before measuring the analyte concentration toallow for diffusion of the redox mediator and/or second electrontransfer agent.

Background Signal

In at least some instances, a diffusible redox mediator may shuttle backand forth from the working electrode to the counter electrode even inthe absence of analyte. This typically creates a background signal. Forcoulometric measurements, this background signal is referred to hereinas “Q_(Back).” The background signal corresponds to the charge passed inan electrochemical assay in the absence of the analyte. The backgroundsignal typically has both a transient component and a steady-statecomponent. At least a portion of the transient component may result, forexample, from the establishment of a concentration gradient of themediator in a particular oxidation state. At least a portion of thesteady-state component may result, for example, from the redox mediatorshuttling between the working electrode and counter or counter/referenceelectrode. Shuttling refers to the redox mediator being electrooxidized(or electroreduced) at the working electrode and then beingelectroreduced (or electrooxidized) at the counter or counter/referenceelectrode, thereby making the redox mediator available to beelectrooxidized (or electroreduced) again at the working electrode sothat the redox mediator is cycling between electrooxidation andelectroreduction.

The amount of shuttling of the redox mediator, and therefore, thesteady-state component of the background signal varies with, forexample, the effective diffusion coefficient of the redox mediator, theviscosity of the sample, the temperature of the sample, the dimensionsof the electrochemical cell, the distance between the working electrodeand the counter or counter/reference electrode, and the angle betweenthe working electrode and the counter or counter/reference electrode.

In some instances, the steady-state component of the background signalmay contain noise associated with (a) variability in, for example, thetemperature of the sample, the sample viscosity, or any other parameteron which the background signal depends during the duration of the assay,or (b) imperfections in the electrochemical cell, such as, for example,non-uniform separation between the working electrode and the counter orcounter/reference electrode, variations in electrode geometry, orprotrusions from the working electrode, the counter electrode, and/orthe counter/reference electrode.

Although the steady-state component of the background signal may bereproducible, any noise inherently is not reproducible. As a result, thenoise adversely affects accuracy. In some cases, the background signaland noise are related. As a result, the noise, and the error itintroduces, can be reduced by reducing the background signal. Forexample, reducing the shuttling of the mediator between the workingelectrode and counter electrode or counter/reference electrode willlikely reduce the noise associated with changes in sample temperatureand viscosity which affect diffusion of the redox mediator.

Thus, to increase the accuracy of the measurements or to decrease errorin the measurements in those instances when reducing a background signalalso reduces noise, a moderate to near-zero level of background signalis desirable. In at least some instances, the sensor is constructed sothat the background signal is at most five times the size of a signalgenerated by electrolysis of an amount of analyte. Preferably, thebackground signal is at most 200%, 100%, 50%, 25%, 10%, or 5% of thesignal generated by electrolysis of the analyte. In the case ofamperometry, this comparison may be made by determining the ratio of thecurrent from the shuttling of the redox mediator to the currentgenerated by the electrolysis of the analyte. In the case ofpotentiometry, this comparison may be made by determining the potentialmeasurement from the shuttling of the redox mediator and the potentialmeasurement generated by electrolysis of the analyte. In the case ofcoulometry, this comparison may be made by determining the chargetransferred at the working electrode by the shuttling of the redoxmediator and the charge transferred at the working electrode by theelectrolysis of the analyte.

The size of the background signal may be compared to a predeterminedamount of analyte. The predetermined amount of analyte in a sample maybe, for example, an expected or average molar amount of analyte. Theexpected or average molar amount of analyte may be determined as, forexample, the average value for users or individuals; an average valuefor a population; a maximum, minimum, or average of a normalphysiological range; a maximum or minimum physiological value for apopulation; a maximum or minimum physiological value for users orindividuals; an average, maximum, or minimum deviation outside a normalphysiological range value for users, individuals, or a population; adeviation above or below an average value for a population; or anaverage, maximum, or minimum deviation above or below an average normalphysiological value for users or individuals. A population may bedefined by, for example, health, sex, or age, such as, for example, anormal adult, child, or newborn population. If a population is definedby health, the population may include people lacking a particularcondition or alternatively, having a particular condition, such as, forexample, diabetes. Reference intervals pertaining to average or expectedvalues, such as, for example, those provided in Tietz Textbook ofClinical Chemistry, Appendix (pp. 2175-2217) (2nd Ed., Carl A. Burtisand Edward R. Ashwood, eds., W.D. Saunders Co., Philadelphia 1994)(incorporated herein by reference) may be used as guidelines, but aphysical examination or blood chemistry determination by a skilledphysician may also be used to determine an average or expected value foran individual. For example, an adult may have glucose in a concentrationof 65 to 95 mg/dL in whole blood or L-lactate in a concentration of 8.1to 15.3 mg/dL in venous whole blood after fasting, according to TietzTextbook of Clinical Chemistry. An average normal physiologicalconcentration for an adult, for example, may then correspond to 80 mg/dLfor glucose or 12.7 mg/dL for lactate. Other examples include a personhaving juvenile onset diabetes, yet good glycemic control, and a glucoseconcentration between about 50 mg/dL and 400 mg/dL, thereby having anaverage molar amount of 225 mg/dL. In another instance, a non-diabeticadult may have a glucose concentration between about 80 mg/dL (afterfasting) and 140 mg/dL (after consuming food), thereby having an averagemolar amount of 110 mg/dL.

Additional analytes that may be determined include, for example, acetylcholine, amylase, bilirubin, cholesterol, chorionic gonadotropin,creatine kinase (e.g., CK-MB), creatine, DNA, fructosamine, glucose,glutamine, growth hormones, hormones, ketones, lactate, peroxide,prostate-specific antigen, prothrombin, RNA, thyroid stimulatinghormone, and troponin. The concentration of drugs, such as, for example,antibiotics (e.g., gentamicin, vancomycin, and the like), digitoxin,digoxin, drugs of abuse, theophylline, and warfarin, may also bedetermined. Assays suitable for determining the concentration of DNAand/or RNA are disclosed in U.S. Pat. No. 6,281,006, U.S. PatentApplication having Ser. No. 09/145,776 and described in U.S. ProvisionalPatent Application Ser. Nos. 60/090,517, 60/093,100, and 60/114,919,incorporated herein by reference.

To construct a sensor having a particular ratio of background signal toanalyte signal from electrolysis, several parameters relating to currentand/or charge from the redox mediator shuttling background signal and/orfrom the signal generated by electrolysis of the analyte may beconsidered and chosen to obtain a desired ratio. Typically, the signaldetermined for a coulometric assay is the charge; whereas the signaldetermined for an amperometric assay is the current at the time when themeasurement is taken. Because the current and charge depend on severalparameters, the desired ratio for background signal generated byshuttling of the redox mediator to signal generated by electrolysis ofthe analyte may be accomplished by a variety of sensor configurationsand methods for operating a sensor.

Controlling Background Signal

One method of controlling background signal includes using a redoxmediator that a) oxidizes the analyte at a half wave potential, asmeasured by cyclic voltammetry in 0.1 M NaCl at pH 7, of no more thanabout +100 mV relative to the potential of a reference orcounter/reference electrode or b) reduces the analyte at a half wavepotential, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, ofno less than about −100 mV relative to the potential of a reference orcounter/reference electrode. A suitable reference or counter/referenceelectrode (e.g., a silver/silver chloride electrode) may be chosen.Preferably, the redox mediator a) oxidizes the analyte at a half wavepotential, as measured by cyclic voltammetry in 0.1 M NaCl at pH 7, ofno more than about +50 mV, +25 mV, 0 mV, −25 mV, −50 mV, −100 mV, or−150 mV relative to the potential of the reference or counter/referenceelectrode or b) reduces the analyte at a half wave potential, asmeasured by cyclic voltammetry in 0.1 M NaCl at pH 7, of no less thanabout −50 mV, −25 mV, 0 mV, +25 mV, +50 mV, +100 mV, +150 mV, or +200 mVrelative to the potential of the reference or counter/referenceelectrode. Alternatively, in the case of reduction of the redox mediatorby the counter electrode, the sensor is operated at an applied potentialof no more than about +100 mV, +50 mV, +25 mV, 0 mV, −25 mV, −50 mV,−100 mV, or −150 mV between the working electrode and the counter orcounter/reference electrode. In the case of oxidation of the redoxmediator at the counter electrode, the sensor is operated at an appliedpotential of no less than about −100 mV, −50 mV, −25 mV, 0 mV, +25 mV,+50 mV, +100 mV, +150 mV, or +200 mV between the working electrode andthe counter or counter/reference electrode.

Another method includes controlling the applied potential such that foran electrooxidative assay the redox mediator is not readily reduced atthe counter or counter/reference electrode or for an electroreductiveassay the redox mediator is not readily oxidized at the counter orcounter/reference electrode. This can be accomplished, for example, inan electrooxidative assay by using a sensor having a diffusible redoxmediator with a potential, relative to a reference or counter/referenceelectrode, that is negative with respect to the potential of the counterelectrode (relative to a reference electrode) or the counter/referenceelectrode. The potential (relative to a reference or counter/referenceelectrode) of the working electrode is chosen to be positive withrespect to the redox mediator and may be negative with respect to thecounter or counter/reference electrode, so that the redox mediator isoxidized at the working electrode. For example, when theelectrooxidation of an analyte is mediated by a diffusible redoxmediator with a potential of −200 mV versus the reference orcounter/reference electrode, and the potential at which the workingelectrode is poised is −150 mV relative to the reference orcounter/reference electrode, then the redox mediator is substantiallyoxidized at the working electrode and will oxidize the analyte. Further,if some of the oxidized redox mediator reaches the counter orcounter/reference electrode, the redox mediator will not be readilyreduced at the counter or counter/reference electrode because thecounter or counter/reference electrode is poised well positive (i.e.,150 mV) of the potential of the redox mediator.

In an electroreductive assay, a sensor is provided having a diffusibleredox mediator with a formal potential, relative to a reference orcounter/reference electrode, that is positive with respect to thepotential of the counter or counter/reference electrode. The potential,relative to the reference or counter/reference electrode, of the workingelectrode is chosen to be negative with respect to the redox mediatorand may be poised positive with respect to the counter orcounter/reference electrode, so that the redox mediator is reduced atthe working electrode.

Still another method of limiting background current includes having theredox mediator become immobilized when reacted on the counter electrodeor counter/reference electrode by, for example, precipitation orpolymerization. For example, the mediator may be cationic in theoxidized state, but neutral and much less soluble in the reduced state.Reduction at the counter/reference electrode leads to precipitation ofthe reduced, neutral mediator on the counter/reference electrode.

Another sensor configuration suitable for controlling background signalincludes a sensor having a molar amount of redox mediator that isstoichiometrically the same as or less than an expected or average molaramount of analyte. The expected or average molar amount of analyte maybe determined as already explained above. The expected or average molaramount of analyte may be determined as, for example, the average valuefor users or individuals; an average value for a population; a maximum,minimum, or average of a normal physiological range; a maximum orminimum physiological value for a population; a maximum or minimumphysiological value for users or individuals; an average, maximum, orminimum deviation outside a normal physiological range value for users,individuals, or a population; a deviation above or below an averagevalue for a population; or an average, maximum, or minimum deviationabove or below an average normal physiological value for users orindividuals. A population may be defined by, for example, health, sex,or age, such as, for example, a normal adult, child, or newbornpopulation. If a population is defined by health, the population mayinclude people lacking a particular condition or alternatively, having aparticular condition, such as, for example, diabetes. Referenceintervals pertaining to average or expected values, such as, forexample, those provided in Tietz Textbook of Clinical Chemistry, supra,may be used as guidelines, but a physical examination or blood chemistrydetermination may also determine an average or expected value. Forexample, the physiological average molar amount of analyte may depend onthe health or age of the person from whom the sample is obtained. Thisdetermination is within the knowledge of a skilled physician.

By reducing the concentration of the redox mediator relative to theconcentration of the analyte, the signal attributable to the analyterelative to the signal attributable to the shuttling of the redoxmediator is increased. In implementation of this method, the molaramount of redox mediator may be no more than 50%, 20%, 10%, or 5%, on astoichiometric basis, of the expected or average molar amount ofanalyte.

The amount of redox mediator used in such a sensor configuration shouldfall within a range. The upper limit of the range may be determinedbased on, for example, the acceptable maximum signal due to shuttling ofthe redox mediator; the design of the electrochemical cell, including,for example, the dimensions of the cell and the position of theelectrodes; the effective diffusion coefficient of the redox mediator;and the length of time needed for the assay. Moreover, the acceptablemaximum signal due to redox mediator shuttling may vary from assay toassay as a result of one or more assay parameters, such as, for example,whether the assay is intended to be qualitative, semi-quantitative, orquantitative; whether small differences in analyte concentration serveas a basis to modify therapy; and the expected concentration of theanalyte.

Although it is advantageous to minimize the amount of redox mediatorused, the range for the acceptable amount of redox mediator doestypically have a lower limit. The minimum amount of redox mediator thatmay be used is the concentration of redox mediator that is necessary toaccomplish the assay within a desirable measurement time period, forexample, no more than about 5 minutes or no more than about 1 minute.The time required to accomplish an assay depends on, for example, thedistance between the working electrode and the counter orcounter/reference electrode, the effective diffusion coefficient of theredox mediator, and the concentration of the analyte. In some instances,for example, when no kinetic limitations are present, i.e., shuttling ofthe redox mediator depends only on diffusion, the minimum concentrationof redox mediator may be determined by the following formula:C _(m)=(d ² C _(A))/D _(m) twhere C_(m) is the minimum concentration of mediator required; d is thedistance between a working electrode and a counter or counter/referenceelectrode in a facing arrangement; C_(A) is the average analyteconcentration in the sample; D_(m) is the effective diffusioncoefficient of the mediator in the sample; and t is the desiredmeasurement time.

For example, when the distance between the facing electrode pair is 50the analyte being measured is 5 mM glucose, the redox mediator effectivediffusion coefficient is 10⁻⁶ cm²/sec and the desirable response time isno more than about 1 minute, then the minimum redox mediatorconcentration is 2.08 mM. Under these conditions the background signalwill be less than the signal from the electrooxidation of the analyte.

Yet another sensor configuration for limiting the background currentgenerated by a diffusible redox mediator includes having a barrier tothe flow of the diffusible mediator to the counter electrode. Thebarrier can be, for example, a film through which the redox mediator cannot diffuse or through which the redox mediator diffuses slowly.Examples of suitable films include polycarbonate, polyvinyl alcohol, andregenerated cellulose or cellulose ester membranes. Alternatively, thebarrier can include charged or polar particles, compounds, or functionalgroups to prevent or reduce the flow of a charged redox mediatorrelative to the flow of a charge neutral or less charged analyte. If theredox mediator is positively charged, as are many of the osmium redoxmediators described below, the barrier can be a positively charged orpolar film, such as a methylated poly(1-vinyl imidazole). If the redoxmediator is negatively charged, the barrier can be a negatively chargedor polar film, such as Nafion®. Examples of suitable polar matricesinclude a bipolar membrane, a membrane having a cationic polymercross-linked with an anionic polymer, and the like. In some instances,the barrier reduces the oxidation or reduction of the diffusible redoxmediator at the counter electrode by at least 25%, 50%, or 90%.

Still another sensor configuration for limiting the background currentincludes a sensor having a redox mediator that is more readily oxidizedor reduced on the working electrode than reduced or oxidized on thecounter electrode. The rate of reaction of the redox mediator at anelectrode can be a function of the material of the electrode. Forexample, some redox mediators may react faster at a carbon electrodethan at a Ag/AgCl electrode. Appropriate selection of the electrodes mayprovide a reaction rate at one electrode that is significantly slowerthan the rate at the other electrode. In some instances, the rate ofoxidation or reduction of the diffusible redox mediator at the counterelectrode is reduced by at least 25%, 50%, or 90%, as compared to theworking electrode. In some instances the rate of reaction for the redoxmediator at the counter or counter/reference electrode is controlled by,for example, choosing a material for the counter or counter/referenceelectrode that would require an overpotential or a potential higher thanthe applied potential to increase the reaction rate at the counter orcounter/reference electrode.

Another sensor configuration for limiting background current includeselements suitable for reducing the diffusion of the redox mediator.Diffusion can be reduced by, for example, using a redox mediator with arelatively low diffusion coefficient or increasing the viscosity of thesample in the measurement zone. In another embodiment, the diffusion ofthe redox mediator may be decreased by choosing a redox mediator withhigh molecular weight, such as, for example, greater than 5,000 daltons,preferably greater than 25,000 daltons, and more preferably greater than100,000 daltons.

Redox Mediators

Although any organic or organometallic redox species can be used as aredox mediator, one type of suitable redox mediator is a transitionmetal compound or complex. Examples of suitable transition metalcompounds or complexes include osmium, ruthenium, iron, and cobaltcompounds or complexes. In these complexes, the transition metal iscoordinatively bound to one or more ligands. The ligands are typicallymono-, di-, tri-, or tetradentate. The most preferred ligands areheterocyclic nitrogen compounds, such as, for example, pyridine and/orimidazole derivatives. Multidentate ligands may include multiplepyridine and/or imidazole rings. Alternatively, metallocene derivatives,such as, for example, ferrocene, can be used.

Suitable redox mediators include osmium or ruthenium transition metalcomplexes with one or more ligands, each ligand having one or morenitrogen-containing heterocycles. Examples of such ligands includepyridine and imidazole rings and ligands having two or more pyridineand/or imidazole rings such as, for example, 2,2″-bipyridine; 2,2′:6′,2″-terpyridine; 1,10-phenanthroline; and ligands having the followingstructures:

and derivatives thereof, wherein R₁ and R₂ are each independentlyhydrogen, hydroxy, alkyl, alkoxy, alkenyl, vinyl, allyl, amido, amino,vinylketone, keto, or sulfur-containing groups.

The term “alkyl” includes a straight or branched saturated aliphatichydrocarbon chain having from 1 to 6 carbon atoms, such as, for example,methyl, ethyl isopropyl methylethyl), butyl, tert-butyl(1,1-dimethylethyl), and the Like. Preferably the hydrocarbon chain hasfrom 1 to 3 carbon atoms.

The term “alkoxy” includes an alkyl as defined above joined to theremainder of the structure by an oxygen atom, such as, for example,methoxy, ethoxy, pr epoxy, isopropoxy (1-methylethoxy), butoxy,tert-butoxy, and the like.

The term “alkenyl” includes an unsaturated aliphatic hydrocarbon chainhaving from 2 to 6 carbon atoms, such as, for example, ethenyl,1-propenyl, 2-propenyl, 1-butenyl, 2-methy-1-propenyl, and the like.Preferably the hydrocarbon chain has from 2 to 3 carbon atoms.

The term “amido” includes groups having a nitrogen atom bonded to thecarbon atom of a carbonyl group and includes groups having the followingformulas:

where R₃ and R₄ are each independently hydrogen, alkyl, alkoxy, oralkenyl.

The term “amino” as used herein includes alkylamino, such asmethylamino, diethylamino, N,N-methylethylamino and the like;alkoxyalkylamino, such as N-(ethoxyethyl)amino,N,N-di(methoxyethyl)amino, N,N-(methoxyethyl)(ethoxyethyl)amino, and thelike; and nitrogen-containing rings, such as piperidino, piperazino,morpholino, and the like.

The term “vinylketone” includes a group having the formula:

wherein R₅, R₆, and R₇ itch independently hydrogen, alkyl, alkoxy, oralkenyl.

The term “keto” includes a group having the formula:

wherein R₈ is hydrogen, alkyl, alkoxy, or alkenyl.

The term “sulfur-containing group” includes mercapto, (such asmethylmercapto, ethylmercapto, and the like), alkoxyalkylmercapto (suchas methoxyethylmercapto and the like), alkylsulfoxide (such asmethylsulfoxide and propylsulfoxide and the like), alkoxyalkylsulfoxide(such as ethoxyethylsulfoxide and the like), alkylsulfone (such asmethylsulfone and propylsulfone and the like), and alkoxyalkylsulfone(such as methoxyethylsulfone and the like). Preferably, thesulfur-containing group is a mercapto group.

Other Suitable redox mediators include osmium or ruthenium transitionmetal complexes with one or more ligands, each ligand having one or morenitrogen-containing heterocycles and each nitrogen-containingheterocycle having a second heteroatom selected from the groupconsisting of nitrogen, oxygen, sulfur and selenium.

Examples of ligands having one or more nitrogen-containing heterocyclesand in which each heterocycle has a second heteroatom include ligandshaving the following structures:

wherein Y₁, Y₂, Y₃, and Y₄ are each independently an oxygen atom, asulfur atom, a selenium atom, or a substituted nitrogen atom having theformula NR₉ wherein R₉ is hydrogen, hydroxy, alkyl, alkoxy, alkenyl,amido, amino, vinylketone, keto, or sulfur-containing group. The terms“alkyl,” “alkoxy,” “alkenyl,” “amido,” “amino,” “vinylketone,” “keto,”and “sulfur-containing group” are as defined above.

Suitable derivatives of these ligands include, for example, the additionof alkyl, alkoxy, alkenyl, vinylester, and amido functional groups toany of the available sites on the heterocyclic ring, including, forexample, on the 4-position (i.e., para to nitrogen) of the pyridinerings or on one of the nitrogen atoms of the imidazole ring.

Suitable derivatives of 2,2′-bipyridine for complexation with the osmiumcation include, for example, mono-, di-, and polyalkyl-2,2′-bipyridines,such as 4,4′-dimethyl-2,2′-bipyridine; mono-, di-, andpolyalkoxy-2,2′-bipyridines, such as 4,4′-dimethoxy-2,2′-bipyridine and2,6-dimethoxy-2,2′-bipyridine; mono-, di-, andpolyacetamido-2,2″-bipyridines, such as4,4′-di(acetamido)-2,2′-bipyridine; mono-, di-, andpolyalkylaminoalkoxy-2,2′-bipyridines, such as4,4′-di(N,N-dimethylaminoethoxy)-2,2′-bipyridine; and substituted mono-,di-, and polypyrazolyl-2,2′-bipyridines, such as4,4′-dimethoxy-6-(N-pyrazolyl)-2,2′-bipyridine and4,4′-dimethoxy-6-(N-pyrazolyl ethyl)-2,2-bipyridine.

Suitable derivatives of 1,10-phenanthroline for complexation with theosmium cation include, for example, mono-, di-, andpolyalkyl-1,10-phenanthrolines, such as4,7-dimethyl-1,10-phenanthroline, and mono, di-, andpolyalkoxy-1,10-phenanthrolines, such as4,7-dimethoxy-1,10-phenanthroline and 5-methoxy-1,10-phenanthroline.

Suitable derivatives for 2,2′:6′,2″-terpyridine include, for example,mono-, tri- and polyalkyl-2,2′:6′,2″-terpyridines, such as4,4′,4″-trimethyl-2,2′:6′,2″-terpyridine,4,4′,4″-triethyl-2,2′:6′,2″-terpyridine, and mono-, di-, tri-, andpolyalkoxy-2,2′;6′,2″-terpyridines, such as4,4″-trimethoxy-2,2′:6′,2″-terpyridine and4′-methoxy-2,2′:6′,2″-terpyridine, and mono-, di-, tri-, andpolyamino-2,2′:6′,2″-terpyridine, such as4′-amino-2,2′:6′,2″-terpyridine, and mono-, di-, tri-, andpolyalkylamino-2,2′:6′,2″-terpyridine, such as4′-dimethylamino-2,2′,6′,2″-terpyridine, and mono-, di-, tri-, andpolyalkylthio-2,2′:6,2″-terpyridine, such as4′-methylthio-2,2′:6′,2″-terpyridine and4-methylthio-4′-ethylthio-2,2′:6′,2″-terpyridine.

Suitable derivatives for pyridine include, for example, mono-, di-,tri-, and polysubstituted pyridines, such as2,6-bis(N-pyrazolyl)pyridine, 2,6-bis(3-methyl-N-pyrazolyl)pyridine,2,6-bis(2-imidazolyl)pyridine, 2,6-bis(1-methyl-2-imidazolyl)bipyridine,and 2,6-bis(1-vinyl-2-imidazolyl)pyridine, and mono-, di-, tri-, andpolyaminopyridines, such as 4-aminopyridine 4,4′-diaminobipyridine,4,4′-di(dimethylamino)bipyridine, and 4,4′,4″-triamino terpyridine.

Other suitable derivatives include compounds comprising threeheterocyclic rings. For example, one suitable derivative includes acompound of the formula:

wherein R₁₀, R₁₁, and R₁₂ are each independently hydrogen, hydroxy,alkyl, alkoxy, alkenyl, vinyl, allyl, amide, amino, vinylketone, keto,or sulfur-containing group.

The terms “alkyl,” “alkoxy,” “alkenyl,” “amido,” “amino,” “vinylketone,”“keto,” and “sulfur-containing group” are as defined above.

Other suitable redox mediator derivatives include compounds of theformula:

wherein R₁₃ is hydrogen, hydroxy, alkyl, alkoxy, alkenyl, vinyl, allyl,vinylketone, keto, amido, amino, or sulfur-containing group; and Y₅ andY₆ are each independently a nitrogen or carbon atom.

The terms “alkyl,” “alkoxy,” “alkenyl,” “amido,” “amino,” “vinylketone,”“keto,” and “sulfur-containing group” are as defined above.

Still other, suitable derivatives include compounds of the

wherein R₁₄ is as defined above and Y₇ and Y₈ are each independently asulfur or oxygen atom.

Examples of suitable redox mediators also include, for example, osmiumcations complexed with (a) two bidentate ligands, such as2,2′-bipyridine, 1,10-phenanthroline, or derivatives thereof (the twoligands not necessarily being the same), (h) one tridentate ligand, suchas 2,2′,2″-terpyridine and 2,6-di(imidazol-2-yl)-pyridine, or (c) onebidentate ligand and one tridentate ligand. Suitable osmium transitionmetal complexes include, for example, [(bpy)₂OsLX]^(+/2+),[(dimet)₂OsLX]^(+/2+), [(dmo)₂OsLX]^(+/2+), [terOsLX₂]^(0/+),[trimetOsLX₂]^(0/+), and [(ter)(bpy)LOs]^(2+/3+) where bpy is2,2″-bipyridine, dimet is 4,4′-dimethyl-2,2′-bipyridine, dmo is4,4′-dimethoxy-2,2′-bipyridine, ter is 2,2′:6′,2″-terpyridine, trimet is4,4′,4″-trimethyl-2,2′:6′,2″-terpyridine, L is a nitrogen-containingheterocyclic ligand, and X is a halogen, such as fluorine, chlorine, orbromine.

The redox mediators often exchange electrons rapidly with each other andwith the electrode so that the complex can be rapidly oxidized and/orreduced. In general, iron complexes are more oxidizing than rutheniumcomplexes, which, in turn, are more oxidizing than osmium complexes. Inaddition, the redox potential generally increases with the number ofcoordinating heterocyclic rings; six-membered heterocyclic ringsincrease the potential more than five membered rings, except when thenitrogen coordinating the metal is formally an anion. This is the caseonly if the nitrogen in the ring is bound to both of its neighboringcarbon atoms by single bonds. If the nitrogen is formally an anion thenthe redox potential generally increases more upon coordination of themetal ion.

At least some diffusible redox mediators include one or more pyridine orimidazole functional groups. The imidazole functional group can alsoinclude other substituents and can be, for example, vinyl imidazole,e.g., 1-vinyl imidazole, or methylimidazole, e.g., 1-methylimidazole.Examples of suitable diffusible mediators may include [Os(dmo)₂(1-vinylimidazole)X]X, [Os(dmo)₂(1-vinyl imidazole)X]X₂,[Os(dmo)₂(imidazole)X]X, [Os(dmo)₂(imidazole)X]X₂,[Os(dmo)₂(1-methylimidazole)X]X₂, and [Os(dmo)₂(methylimidazole)X]X₂,where dmo is 4,4-dimethoxy-2,2′-bipyridine, and X is halogen asdescribed above.

Other osmium-containing redox mediators include[Os((methoxy)₂phenanthroline)₂(N-methylimidazole)X]^(+/2+);[Os((acetamido)₂bipyridine)₂(L)X]^(+/2+), where L is a monodentatenitrogen-containing compound (including, but not limited to, animidazole derivative) chosen to refine the potential; andOs(terpyridine)(L)₂Cl, where L is an aminopyridine, such as adialkylaminopyridine; an N-substituted imidazole, such as N-methylimidazole; an oxazole; a thiazole; or an alkoxypyridine, such asmethoxypyridine. X is halogen as described above.

Osmium-free diffusible redox mediators include, for example,phenoxazines, such as, 7-dimethylamino-1,2-benzophenoxazine (MeldolaBlue), 1,2-benzophenoxazine, and Nile Blue; 3-β-naphthoyl (BrilliantCresyl Blue); tetramethylphenylenediamine (TMPD);dichlorophenolindophenol (DCIP); N-methyl phenazonium salts, forexample, phenazine methosulfate (PMS), N-methylphenazine methosulfateand methoxyphenazine methosulfate; tetrazolium salts, for example,tetrazolium blue or nitrotetrazolium blue; and phenothiazines, forexample, toluidine blue O.

Examples of other redox species include stable quinones and species thatin their oxidized state have quinoid structures, such as Nile Blue andindophenol. Examples of suitable quinones include, for example,derivatives of naphthoquinone, phenoquinone, benzoquinone,naphthenequinone, and the like. Examples of naphthoquinone derivativesinclude juglone (i.e., 5-hydroxy-1,4-naphthoquinone) and derivativesthereof, such as, for example,2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone,2,3-dimethyl-5,8-dihydroxy-1,4-naphthoquinone,2-chloro-5,8-dihydroxy-1,4-naphthoquinone,2,3-methoxy-5-hydroxy-1,4-naphthoquinone, and the like. Other examplesinclude aminonaphthoquinones, such as, for example,morpholino-naphthoquinones, such as2-chloro-3-morpholino-1,4-naphthoquinone; piperidino-naphthoquinones,such as 2-methyl-3-peperidino-1,4-naphthoquinone;piperazino-naphthoquinones, such as2-ethoxy-3-piperazino-1,4-naphthoquinone; and the like.

Suitable phenoquinone derivatives include, for example, coerulignone3,3′,5,5′-tetramethoxydiphenoquinone) and derivatives thereof, such as,for example, 3,3′,5,5′-tetramethyldiphenoquinone,3,3′,5,5′-tetrahydroxydiphenoquinone, and the like.

Suitable benzoquinone derivatives include, for example, coenzyme Q₀(i.e., 2,3-dimethoxy-5-methyl-1,4-benzoquinone) and derivatives thereof,such as, for example, 2,3,5-trimethyl-1,4-benzoquinone,2,3-dimethyl-5-methoxy-1,4-benzoquinone,2,3-dimethyl-5-hydroxy-1,4-benzoquinone, and the like.

Other suitable quinone derivatives include, for example,acenaphthenequinone and ubiquinones, such as, for example, coenzyme Q,including Q₁, Q₂, Q₆, Q₇, Q₉, and Q₁₀.

Still other suitable osmium-free diffusible redox mediators include, forexample, Taylor's blue (i.e., 1,9-dimethylmethylene blue),N,N′-diethylthiacyanine iodide, and thionine.

In another method, a sensing layer 32 contains a non-leachable (i.e.,non-releasable) redox mediator and is disposed on a portion of theworking electrode 22. The non-leachable redox mediator can be, forexample, a redox polymer (i.e., a polymer having one or more redoxspecies). Preferably, there is little or no leaching of thenon-leachable redox mediator away from the working electrode 22 into thesample during the measurement period, which is typically less than about5 minutes. The redox mediators of this embodiment can be bound orotherwise immobilized on the working electrode 22 to prevent leaching ofthe mediator into the sample. The redox mediator can be bound orotherwise immobilized on the working electrode by known methods, forexample, formation of multiple ion bridges with a counterchargedpolyelectrolyte, covalent attachment of the redox mediator to a polymeron the working electrode, entrapment of the redox mediator in a matrixthat has a high affinity for the redox mediator, or bioconjugation ofthe redox mediator with a compound bound to the working electrode. Inone embodiment, a cationic exchange membrane may be used to entrap ananionic redox compound. Similarly, in another embodiment, an anionicexchange membrane may be used to entrap a cationic redox compound. Instill another embodiment involving bioconjugation, a biotin-bound redoxmediator can conjugate with avidin or straptavidin in a matrix near orimmobilized on the working electrode. Still another embodiment includeshaving a digoxin or digoxigenin redox mediator react with antidigoxin ina matrix near or immobilized on a working electrode.

Preferred non-leachable redox mediators are redox polymers, such aspolymeric transition metal compounds or complexes. Typically, thepolymers used to form a redox polymer have nitrogen-containingheterocycles, such as pyridine, imidazole, or derivatives thereof forbinding as ligands to the redox species. Suitable polymers forcomplexation with redox species, such as the transition metal complexes,described above, include, for example, polymers and copolymers ofpoly(1-vinyl imidazole) (referred to as “PVI”) and poly(4-vinylpyridine) (referred to as “PVP”), as well as polymers and copolymers ofpoly(acrylic acid) or polyacrylamide that have been modified by theaddition of pendant nitrogen-containing heterocycles, such as pyridineand imidazole. Modification of poly(acrylic acid) may be performed byreaction of at least a portion of the carboxylic acid functionalitieswith an aminoalkylpyridine or aminoalkylimidazole, such as4-ethylaminopyridine, to form amides. Suitable copolymer substituents ofPVI, PVP, and poly(acrylic acid) include acrylonitrile, acrylamide,acrylhydrazide, and substituted or quaternized 1-vinyl imidazole. Thecopolymers can be random or block copolymers.

The transition metal complexes of non-leachable redox polymers aretypically covalently or coordinatively bound with thenitrogen-containing heterocycles (e.g., imidazole and/or pyridine rings)of the polymer. The transition metal complexes may have vinyl functionalgroups through which the complexes can be co-polymerized. Suitable vinylfunctional groups include, for example, vinylic heterocycles, amides,nitriles, carboxylic acids, sulfonic acids, or other polar vinyliccompounds. An example of a redox polymer of this type is poly(vinylferrocene) or a derivative of poly(vinyl ferrocene) functionalized toincrease swelling of the redox polymer in water.

Another type of redox polymer contains an ionically-bound redox species,by forming multiple ion-bridges. Typically, this type of mediatorincludes a charged polymer coupled to an oppositely charged redoxspecies. Examples of this type of redox polymer include a negativelycharged polymer such as Nafion® (DuPont) coupled to multiple positivelycharged redox species such as an osmium or ruthenium polypyridyl cation.Another example of an ionically-bound mediator is a positively chargedpolymer such as quaternized poly(4-vinyl pyridine) or poly(1-vinylimidazole) coupled to a negatively charged redox species such asferricyanide or ferrocyanide. The preferred ionically-bound redoxspecies is a multiply charged, often polyanionic, redox species boundwithin an oppositely charged polymer.

Another suitable redox polymer includes a redox species coordinativelybound to a polymer. For example, the mediator may be formed bycoordination of an osmium, ruthenium, or cobalt 2,2′-bipyridyl complexto poly(1-vinyl imidazole) or poly(4-vinyl pyridine) or byco-polymerization of, for example, a 4-vinyl-2,2′-bipyridyl osmium,ruthenium, or cobalt complex with 1-vinyl imidazole or 4-vinyl pyridine.

Typically, the ratio of osmium or ruthenium transition metal complexesto imidazole and/or pyridine groups of the non-leachable redox polymersranges from 1:20 to 1:1, preferably from 1:15 to 1:2, and morepreferably from 1:10 to 1:4. Generally, the redox potentials depend, atleast in part, on the polymer with the order of redox potentials beingpoly(acrylic acid)<PVI<PVP.

A variety of methods may be used to immobilize a redox polymer on anelectrode surface. One method is adsorptive immobilization. This methodis particularly useful for redox polymers with relatively high molecularweights. The molecular weight of a polymer may be increased, forexample, by cross-linking. The polymer of the redox polymer may containfunctional groups, such as, for example, hydrazide, amine, alcohol,heterocyclic nitrogen, vinyl, allyl, and carboxylic acid groups, thatcan be crosslinked using a crosslinking agent. These functional groupsmay be provided on the polymer or one or more of the copolymers.Alternatively or additionally, the functional groups may be added by areaction, such as, for example, quaternization. One example is thequaternization of PVP with bromoethylamine groups.

Suitable cross-linking agents include, for example, molecules having twoor more epoxide (e.g., poly(ethylene glycol) diglycidyl ether (PEGDGE)),aldehyde, aziridine, alkyl halide, and azide functional groups orcombinations thereof. When a polymer has multiple acrylate functions, itcan be crosslinked with a di- or polythiol; when it has multiple thiolfunctions it can be crosslinked with a di- or polyacrylate. Otherexamples of cross-linking agents include compounds that activatecarboxylic acid or other acid functional groups for condensation withamines or other nitrogen compounds. These cross-linking agents includecarbodiimides or compounds with active N-hydroxysuccinimide or imidatefunctional groups. Yet other examples of cross-linking agents arequinones (e.g., tetrachlorobenzoquinone and tetracyanoquinodimethane)and cyanuric chloride. Other cross-linking agents may also be used. Insome embodiments, an additional cross-linking agent is not required.Further discussion and examples of cross-linking and cross-linkingagents are found in U.S. Pat. Nos. 5,262,035; 5,262,305; 5,320,725;5,264,104; 5,264,105; 5,356,786; and 5,593,852, herein incorporated byreference.

In another embodiment, the redox polymer is immobilized by thefunctionalization of the electrode surface and then the chemicalbonding, often covalently, of the redox polymer to the functional groupson the electrode surface. One example of this type of immobilizationbegins with a poly(4-vinyl pyridine). The polymer's pyridine rings are,in part, complexed with a reducible/oxidizable species, such as[Os(bpy)₂Cl]^(+/2+) where bpy is 2,2′-bipyridine. Part of the pyridinerings are quaternized by reaction with 2-bromoethylamine. The polymer isthen crosslinked, for example, using a diepoxide, such as poly(ethyleneglycol) diglycidyl ether.

Carbon surfaces can be modified for attachment of a redox polymer, forexample, by electroreduction of a diazonium salt. As an illustration,reduction of a diazonium salt formed upon diazotization ofp-aminobenzoic acid modifies a carbon surface with phenylcarboxylic acidfunctional groups. These functional groups can be activated by acarbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC). The activated functional groups are bound with anamine-functionalized redox couple, such as, for example, the quaternizedosmium-containing redox polymer described above or2-aminoethylferrocene, to form the redox couple.

Similarly, gold and other metal surfaces can be functionalized by, forexample, an amine, such as cystamine, or by a carboxylic acid, such asthioctic acid. A redox couple, such as, for example,[Os(bpy)₂pyridine-4-carboxylate)Cl]^(0/+), is activated by1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) toform a reactive O-acylisourea which reacts with the gold-bound amine toform an amide. The carboxylic acid functional group of thioctic acid canbe activated with EDC to bind a polymer or protein amine to form anamide.

When the enzyme used is PQQ glucose dehydrogenase or glucose oxidase,the preferred non-leachable redox mediators have a redox potentialbetween about −300 mV to about +400 mV versus the standard calomelelectrode (SCE). The most preferred non-leachable redox mediators haveosmium redox centers and a redox potential more negative than +100 mVversus SCE, more preferably the redox potential is more negative than 0mV versus SCE, and most preferably is near −150 mV versus SCE.

In at least some instances, the redox mediators of the sensors areair-oxidizable. This means that the redox mediator is oxidized by air,preferably, so that at least 90% of the mediator is in an oxidized stateprior to introduction of sample into the sensor. Air-oxidizable redoxmediators include osmium cations complexed with two mono-, di-, orpolyalkoxy-2,2′-bipyridine or mono-, di-, orpolyalkoxy-1,10-phenanthroline ligands, the two ligands not necessarilybeing the same, and further complexed with polymers or other ligandshaving pyridine and imidazole functional groups. In particular,Os[4,4′-dimethoxy-2,2′-bipyridine]₂Cl^(+/+2) complexed with poly(4-vinylpyridine) or poly(1-vinyl imidazole) attains approximately 90% or moreoxidation in air. The air oxidation of the redox mediator may take placewhile the redox mediator is a solid, such as, for example, when it iscoated on the sensor in a dry state and stored. Alternatively, the airoxidation of the redox mediator may take place while the redox mediatoris in solution, such as, for example, prior to the solution beingapplied onto the sensor and dried. In the case in which the redoxmediator is air oxidized in solution, the solution containing the redoxmediator may be kept in storage for a period of time sufficient to airoxidize the mediator prior to use of the solution in the manufacturingprocess.

Second Electron Transfer Agent

In a preferred embodiment of the invention, the sensor includes a redoxmediator and a second electron transfer agent which is capable oftransferring electrons to or from the redox mediator and the analyte.The second electron transfer agent may be diffusible or may benon-leachable (e.g., entrapped in or coordinatively, covalently, orionically bound to the redox polymer). One example of a suitable secondelectron transfer agent is an enzyme which catalyzes a reaction of theanalyte. For example, a glucose oxidase or glucose dehydrogenase, suchas pyrroloquinoline quinone glucose dehydrogenase (PQQ), is used whenthe analyte is glucose. A lactate oxidase fills this role when theanalyte is lactate. Other enzymes can be used for other analytes. Theseenzymes catalyze the electrolysis of an analyte by transferringelectrons between the analyte and the electrode via the redox mediator.In some embodiments, the second electron transfer agent isnon-leachable, and more preferably immobilized on the working electrode,to prevent unwanted leaching of the agent into the sample. This isaccomplished, for example, by cross-linking the non-leachable secondelectron transfer agent with the non-leachable redox mediator, therebyproviding a sensing layer with non-leachable components on the workingelectrode. In other embodiments, the second electron transfer agent isdiffusible (and may be disposed on any surface of the sample chamber orplaced in the sample).

Counter Electrode

Counter electrode 24, as illustrated in FIGS. 1-4, may be constructed ina manner similar to working electrode 22. Counter electrode 24 may alsobe a counter/reference electrode. Alternatively, a separate referenceelectrode may be provided in contact with the sample chamber. Suitablematerials for the counter/reference or reference electrode includeAg/AgCl or Ag/AgBr printed on a non-conducting base material or silverchloride on a silver metal base. The same materials and methods may beused to make the counter electrode as are available for constructing theworking electrode 22, although different materials and methods may alsobe used. A tab 25 may be provided on the electrode for convenientconnection to the external electronics (not shown), such as acoulometer, potentiostat, or other measuring device.

Electrode Configuration

In one embodiment of the invention, working electrode 22 and counterelectrode 24 are disposed opposite to and facing each other to form afacing electrode pair as depicted in FIGS. 1 and 3. In this preferredconfiguration, the sample chamber 26 is typically disposed between thetwo electrodes. For this facing electrode configuration, it is preferredthat the electrodes are separated by a distance of no more than about0.2 mm (i.e., at least one portion of the working electrode is separatedfrom one portion of the counter electrode by no more than 200 μm),preferably no more than 100 μm, and most preferably no more than 50 μm.

The electrodes need not be directly opposing each other; they may beslightly offset. Furthermore, the two electrodes need not be the samesize. Preferably, the counter electrode 24 is at least as large as theworking surface of the working electrode 22. The counter electrode 22can also be formed with tines in a comb shape. Other configurations ofboth the counter electrode and working electrode are within the scope ofthe invention. However, for this particular embodiment, the separationdistance between at least one portion of the working electrode and someportion of the counter electrode preferably does not exceed the limitsspecified hereinabove.

FIGS. 11A, 11B, and 11C illustrate different embodiments of pairs offacing electrodes 22, 24, as described above. A region 21 of overlapbetween the two electrodes 22, 24 typically corresponds to themeasurement zone in which the sample will be interrogated. Each of theelectrodes 22, 24 is a conducting surface and acts as a plate of acapacitor. The measurement zone between the electrodes 22, 24 acts as adielectric layer between the plates. Thus, there is a capacitancebetween the two electrodes 22, 24. This capacitance is a function of thesize of the overlapping electrodes 22, 24, the separation between theelectrodes 22, 24, and the dielectric constant of the material betweenthe electrodes 22, 24. Thus, if the size of the region 21 of theoverlapping electrodes 22, 24 and the dielectric constant of thematerial between the electrodes (e.g., air or a sorbent material) areknown, then the separation between the electrodes can be calculated todetermine the volume of the measurement zone.

FIG. 11A illustrates one embodiment of the invention in which theelectrodes 22, 24 are positioned in a facing arrangement. For thecapacitance to be uniform among similarly constructed analyte sensorshaving this particular sensor configuration, the registration (i.e., thepositioning of the two electrodes relative to one another) should beuniform. If the position of either of the electrodes is shifted in thex-y plane from the position shown in FIG. 11A, the size of the overlap,and therefore, of the capacitance, will change. The same principle holdsfor the volume of the measurement zone.

FIGS. 11B and 11C illustrate other embodiments of the invention withelectrodes 22, 24 in a facing arrangement. In these particulararrangements, the position of either of the electrodes may be shifted,by at least some minimum distance, in the x-y plane relative to theother electrode without a change in the capacitance or the volume of themeasurement zone. In these electrode arrangements, each electrode 22, 24includes an arm 122, 124, respectively, which overlaps with thecorresponding arm of the other electrode. The two arms 122, 124 are notparallel to each other (such as illustrated in FIG. 11A); rather, thearms 122, 124 are disposed at an angle 123, which is greater than zero,relative to each other. In addition, the two arms 122, 124 extend beyondthe region 21 of overlap (i.e., each arm has extra length correspondingto the difference between the length of the arm 222, 224, respectively,and the width 121 of the overlap 21). With these electrode arrangements,there can be a certain amount of allowed imprecision in the registrationof the electrodes 22, 24 which does not change the capacitance of theelectrode arrangement. A desired amount of allowed imprecision in theregistration can be designed into the electrode arrangement by varyingthe angle 123 at which the arms 122, 124 overlap and the size of theextra length of each arm 122, 124 relative to the width 121 of theregion 21 of overlap. Typically, the closer that the arms 122, 124 areto being perpendicular (i.e., angle 123 is 90°, the greater the allowedimprecision. Also, the greater the extra length of each arm 122, 124(which may both be the same length or different lengths) relative to thewidth 121 of the region 21 of overlap, the greater the allowedimprecision. Conversely, the greater the amount of allowed imprecision,the larger the size of the electrodes (for a given electrode width,thickness, and angle 123 of intersection with the other electrode).Thus, the minimum distance that one electrode can be shifted relative tothe other is balanced against the amount of material needed for theelectrodes. Typically, the angle 123 of intersection ranges from 5 to 90degrees, preferably, 30 to 90 degrees, and more preferably 60 to 90degrees. Typically, the ratio of the extra length of an arm 122, 124(corresponding to the difference between the arm length 222, 224 and thewidth 121 of the region 21 of overlap) versus the width 121 of theregion 21 of overlap ranges from 0.1:1 to 50:1, preferably 1:1 to 15:1,and more preferably 4:1 to 10:1.

In another embodiment of the invention, the two electrodes 22, 24 arecoplanar as shown in FIG. 2. In this case, the sample chamber 26 is incontact with both electrodes and is bounded on the side opposite theelectrodes by a non-conducting inert base 30. Suitable materials for theinert base include non-conducting materials such as polyester.

Other configurations of the inventive sensors are also possible. Forexample, the two electrodes may be formed on surfaces that make an angleto each other. One such configuration would have the electrodes onsurfaces that form aright angle. Another possible configuration has theelectrodes on a curved surface such as the interior of a tube. Theworking and counter electrodes may be arranged on that they face eachother from opposite sides of the tube. This is another example of afacing electrode pair. Alternatively, the electrodes may be placed neareach other on the tube wall (e.g., one on top of the other orside-by-side).

In any configuration, the two electrodes must be configured so that theydo not make direct electrical contact with each other, to preventshorting of the electrochemical sensor. This may be difficult to avoidwhen the facing electrodes are separated, over the average, by no morethan about 100 μm.

A spacer 28 can be used to keep the electrodes apart when the electrodesface each other as depicted in FIGS. 1 and 3. The spacer is typicallyconstructed from an inert non-conducting material such aspressure-sensitive adhesive, polyester, Mylar™, Kevlar™ or any otherstrong, thin polymer film, or, alternatively, a thin polymer film suchas a Teflon™ film, chosen for its chemical inertness. In addition topreventing contact between the electrodes, the spacer 28 often functionsas a portion of the boundary for the sample chamber 26 as shown in FIGS.1-4. Other spacers include layers of adhesive and double-sided adhesivetape (e.g., a carrier film with adhesive on opposing sides of the film).

Sample Chamber

The sample chamber 26 is typically defined by a combination of theelectrodes 22, 24, an inert base 30, and a spacer 28 as shown in FIGS.1-4. A measurement zone is contained within this sample chamber and isthe region of the sample chamber that contains only that portion of thesample that is interrogated during the analyte assay. In the embodimentof the invention illustrated in FIGS. 1 and 2, sample chamber 26 is thespace between the two electrodes 22, 24 and/or the inert base 30. Inthis embodiment, the sample chamber has a volume that is preferably nomore than about 1 μL, more preferably no more than about 0.5 μL, andmost preferably no more than about 0.25 μL. In the embodiment of theinvention depicted in FIGS. 1 and 2, the measurement zone has a volumethat is approximately equal to the volume of the sample chamber. In apreferred embodiment the measurement zone includes 80% of the samplechamber, 90% in a more preferred embodiment, and about 100% in a mostpreferred embodiment.

In another embodiment of the invention, shown in FIG. 3, sample chamber26 includes much more space than the region proximate electrodes 22, 24.This configuration makes it possible to provide multiple electrodes incontact with one or more sample chambers, as shown in FIG. 5. In thisembodiment, sample chamber 26 is preferably sized to contain a volume ofno more than about 1 μL, more preferably no more than about 0.5 μL, andmost preferably no more than about 0.25 μL. The measurement zone (i.e.,the region containing the volume of sample to be interrogated) isgenerally sized to contain a volume of sample of no more than about 1μL, preferably no more than about 0.5 μL, more preferably no more thanabout 0.25 μL, and most preferably no more than about 0.1 μL. Oneparticularly useful configuration of this embodiment positions workingelectrode 22 and counter electrode 24 facing each other, as shown inFIG. 3. In this embodiment, the measurement zone, corresponding to theregion containing the portion of the sample which will be interrogated,is the portion of sample chamber 26 bounded by the working surface ofthe working electrode and disposed between the two facing electrodes.

In both of the embodiments discussed above, the thickness of the samplechamber and of the measurement zone correspond typically to thethickness of spacer 28 (e.g., the distance between the electrodes inFIGS. 1 and 3, or the distance between the electrodes and the inert basein FIG. 2). The spacer may be, for example, an adhesive or double-sidedadhesive tape or film. Preferably, this thickness is small to promoterapid electrolysis of the analyte, as more of the sample will be incontact with the electrode surface for a given sample volume. Inaddition, a thin sample chamber helps to reduce errors from diffusion ofanalyte into the measurement zone from other portions of the samplechamber during the analyte assay, because diffusion time is longrelative to the measurement time. Typically, the thickness of the samplechamber is no more than about 0.2 mm. Preferably, the thickness of thesample chamber is no more than about 0.1 mm and, more preferably, thethickness of the sample chamber is about 0.05 mm or less.

The sample chamber may be formed by other methods. Exemplary methodsinclude embossing, indenting, or otherwise forming a recess in asubstrate within which either the working electrode 22 or counterelectrode 24 is formed. FIGS. 12A and 12B illustrate one embodiment ofthis structure. First, a conducting layer 100 is formed on an inertnon-conducting base material 102. As described above, the conductinglayer 100 can include gold, carbon, platinum, ruthenium dioxide,palladium, or other non-corroding materials. The inert non-conductingbase material 102 can be made using a polyester, other polymers, orother non-conducting, deformable materials. A recess 104 is then formedin a region of the non-conducting base material 102 so that at least aportion of the conducting layer 100 is included in the recess 104. Therecess 104 may be formed using a variety of techniques includingindenting, deforming, or otherwise pushing in the base material 102. Oneadditional exemplary method for forming the recess includes embossingthe base material 102. For example, the base material 102 may be broughtinto contact with an embossing roll or stamp having raised portions,such as punch members or channels, to form the recess 104. In someembodiments, the base material 102 may be heated to soften the material.

The recess 104 may be circular, oval, rectangular, or any other regularor irregular shape. Alternatively, the recess 104 may be formed as achannel which extends along a portion of the base material 102. Theconducting layer 100 may extend along the entire channel or only aportion of the channel. The measurement zone may be restricted to aparticular region within the channel by, for example, depositing thesensing layer 32 on only that portion of the conducting layer 100 withinthe particular region of the channel. Alternatively, the measurementzone may be defined by placing a second electrode 107 over only thedesired region of the first electrode 105.

At least a portion, and in some cases, all, of the conducting layer 100is situated in the recess 104. This portion of the conducting layer 100may act as a first electrode 105 (a counter electrode or a workingelectrode). If the conducting layer 100 forms the working electrode,then a sensing layer 32 may be formed over a portion of the conductinglayer 100 by depositing a non-leachable redox mediator and/or secondelectron transfer agent in the recess 104, as shown in FIG. 12B. If adiffusible redox mediator or second electron transfer agent is used,then the diffusible material may be disposed on any surface in thesample chamber or in the sample.

A second electrode 107 is then formed by depositing a second conductinglayer on a second base material 106. This second electrode 107 is thenpositioned over the first electrode 105 in a facing arrangement.Although not illustrated, if the redox mediator is non-leachable it willbe understood that if the first electrode 105 were to function as acounter electrode, then the sensing layer 32 would be deposited on thesecond electrode 107 which would then function as the working electrode.If the redox mediator is diffusible, however, the redox mediator may bedisposed on any surface of the sample chamber or may be placed in thesample.

In one embodiment, the second base material 106 rests on a portion ofthe first base material 102 and/or the conducting layer 100 which is notdepressed, so that the second electrode 107 extends into the recess. Inanother embodiment, there is a spacer (not shown) between the first andsecond base materials 102, 106. In this embodiment, the second electrode107 may or may not extend into the recess. In any case, the first andsecond electrodes 105, 107 do not make contact, otherwise the twoelectrodes would be shorted.

The depth of the recess 104 and the volume of the conductive layer 100,sensing layer 32, and the portion, if any, of the second electrode 107in the recess 104 determines the volume of the measurement zone. Thus,the predictability of the volume of the measurement zone relies on theextent to which the formation of the recess 104 is uniform.

In addition to the conducting layer 100, a sorbent layer 103, describedin detail below, may be deposited on the base material 102 prior toforming the recess 104, as shown in FIG. 14A. The sorbent material 103may be indented, embossed, or otherwise deformed with the conductinglayer 100 and base material 102, as shown in FIG. 14B. Alternatively,the sorbent material 103 may be deposited after the conducting layer 100and base material 102 are indented, embossed, or otherwise deformed tomake the recess 104.

In another exemplary method for forming the analyte sensor, a recess 114is formed in a first base material 112, as shown in FIGS. 13A and 13B.The recess may be formed by indenting, embossing, etching (e.g., usingphotolithographic methods or laser removal of a portion of the basematerial), or otherwise deforming or removing a portion of the basematerial 112. Then a first conducting layer 110 is formed in the recess114. Any of the conductive materials discussed above may be used. Apreferred material is a conductive ink, such as a conductive carbon inkavailable, for example, from Ercon, Inc. (Wareham, Mass.). Theconductive ink typically contains metal or carbon dissolved or dispersedin a solvent or dispersant. When the solvent or dispersant is removed,the metal or carbon forms a conductive layer 110 that can then be usedas a first electrode 115. A second electrode 117 can be formed on asecond base material 116 and positioned over the recess 114, asdescribed above. In embodiments having a non-leachable redox mediator, asensing layer 32 is formed on the first electrode 115 to form a workingelectrode, as shown in FIG. 13B. In other embodiments having anon-leachable redox mediator, the sensing layer 32 may be formed on thesecond electrode 117 to form a working electrode. Alternatively, if adiffusible redox mediator is used, then the working electrode need notinclude the sensing layer disposed thereon. In fact, no sensing layer isrequired because the redox mediator may be placed in the sample andlikewise for a diffusible second electron transfer agent, if one ispresent. Any diffusible components may be independently disposed on anysurface of the sample chamber or placed in the sample. Furthermore, asorbent material (not shown) may be formed within the recess, forexample, on the first electrode 115.

A binder, such as a polyurethane resin, cellulose derivative, elastomer(e.g., silicones, polymeric dienes, or acrylonitrile-butadiene-styrene(ABS) resins), highly fluorinated polymer, or the like, may also beincluded in the conductive ink. Curing the binder may increase theconductivity of the conductive layer 110, however, curing is notnecessary. The method of curing the binder may depend on the nature ofthe particular binder that is used. Some binders are cured by heatand/or ultraviolet light.

These structures allow for the formation of electrochemical sensors inwhich the volume of the measurement zone depends, at least in part, onthe accuracy and reproducibility of the recess 104. Embossing, laseretching, photolithographic etching and other methods can be used to makereproducible recesses 104, even on the scale of 200 μm or less.

Sorbent Material

The sample chamber may be empty before the sample is placed in thechamber. Alternatively, the sample chamber may include a sorbentmaterial 34 to sorb and hold a fluid sample during the measurementprocess. Suitable sorbent materials include polyester, nylon, cellulose,and cellulose derivatives such as nitrocellulose. The sorbent materialfacilitates the uptake of small volume samples by a wicking action whichmay complement or, preferably, replace any capillary action of thesample chamber. In addition or alternatively, a portion or the entiretyof the wall of the sample chamber may be covered by a surfactant, suchas, for example, Zonyl FSO.

In some embodiments, the sorbent material is deposited using a liquid orslurry in which the sorbent material is dissolved or dispersed. Thesolvent or dispersant in the liquid or slurry may then be driven off byheating or evaporation processes. Suitable sorbent materials include,for example, cellulose or nylon powders dissolved or dispersed in asuitable solvent or dispersant, such as water. The particular solvent ordispersant should also be compatible with the material of the workingelectrode 22 (e.g., the solvent or dispersant should not dissolve theelectrode).

One of the most important functions of the sorbent material is to reducethe volume of fluid needed to fill the sample chamber and correspondingmeasurement zone of the sensor. The actual volume of sample within themeasurement zone is partially determined by the amount of void spacewithin the sorbent material. Typically, suitable sorbents consist ofabout 5% to about 50% void space. Preferably, the sorbent materialconsists of about 10% to about 25% void space.

The displacement of fluid by the sorbent material is advantageous. Byaddition of a sorbent, less sample is needed to fill sample chamber 26.This reduces the volume of sample that is required to obtain ameasurement and also reduces the time required to electrolyze thesample.

The sorbent material 34 may include a tab 33 which is made of the samematerial as the sorbent and which extends from the sensor, or from anopening in the sensor, so that a sample may be brought into contact withtab 33, sorbed by the tab, and conveyed into the sample chamber 26 bythe wicking action of the sorbent material 34. This provides a preferredmethod for directing the sample into the sample chamber 26. For example,the sensor may be brought into contact with a region of an animal(including human) that has been pierced with a lancet to draw blood. Theblood is brought in contact with tab 33 and drawn into sample chamber 26by the wicking action of the sorbent 34. The direct transfer of thesample to the sensor is especially important when the sample is verysmall, such as when the lancet is used to pierce a portion of the animalthat is not heavily supplied with near-surface capillary vessels andfurnishes a blood sample volume of 1 μL or less.

Methods other than the wicking action of a sorbent may be used totransport the sample into the sample chamber or measurement zone.Examples of such methods for transport include the application ofpressure on a sample to push it into the sample chamber, the creation ofa vacuum by a pump or other vacuum-producing method in the samplechamber to pull the sample into the chamber, capillary action due tointerfacial tension of the sample with the walls of a thin samplechamber, as well as the wicking action of a sorbent material.

The sensor can also be used in conjunction with a flowing sample stream.In this configuration, the sample stream is made to flow through asample chamber. The flow is stopped periodically and the concentrationof the analyte is determined by an electrochemical method, such ascoulometry. After the measurement, the flow is resumed, thereby removingthe sample from the sensor. Alternatively, sample may flow through thechamber at a very slow rate, such that all of the analyte iselectrolyzed in transit, yielding a current dependent only upon analyteconcentration and flow rate.

Other filler materials may be used to fill the measurement zone andreduce the sample volume. For example, glass beads can be deposited inthe measurement zone to occupy space. Preferably, these filler materialsare hydrophilic so that the body fluid can easily flow into themeasurement zone. In some cases, such as glass beads with a high surfacearea, these filler materials may also wick the body fluid into themeasurement zone due to their high surface area and hydrophilicity.

The entire sensor assembly is held firmly together to ensure that thesample remains in contact with the electrodes and that the samplechamber and measurement zone maintain the same volume. This is animportant consideration in the coulometric analysis of a sample, wheremeasurement of a defined sample volume is needed. One method of holdingthe sensor together is depicted in FIGS. 1 and 2. Two plates 38 areprovided at opposite ends of the sensor. These plates are typicallyconstructed of non-conducting materials such as plastics. The plates aredesigned so that they can be held together with the sensor between thetwo plates. Suitable holding devices include adhesives, clamps, nuts andbolts, screws, and the like.

Alternative Sensor Designs

FIGS. 18A to 18C illustrate one alternative sensor design for formationof thin film sensors. The sensor includes a first substrate 500 uponwhich a working electrode 502 is formed. The working electrode 502includes a contact region 503 for connection with external electronics.A spacer 504 (FIG. 18B), such as, for example, a layer of adhesive or adouble-sided tape defines a channel 506 to produce a sample chamber forthe sensor. Two counter (or counter/reference) electrodes 510, 512 areformed on a second substrate 508, as shown in FIG. 18C (inverted withrespect to FIGS. 18A and 18B to show the electrode side up). Thismultiple counter electrode arrangement may provide a fill indicatorfunction, as described below. Each counter electrode 510, 512 has acontact region 511, 513 for connection with external electronics. Thesecond substrate 508 is inverted and placed over the first substrate500, with the spacer 504 between, so that the working electrode 502 andthe two counter electrodes 510, 512 are facing in the region of thechannel 506.

In some instances, the counter electrode 510 nearest an entrance 514 ofthe channel 506 has a surface area within the sample chamber that is atleast two times larger than the other counter electrode 512, and may beat least five or ten times larger. The non-leachable or diffusible redoxmediator and/or second electron transfer agent can be provided on eitherthe first or second substrates 500, 508 in a region corresponding to thechannel 506, as described above.

The working electrode and counter electrodes can be formed to cover theentire channel region (except for a small space between the two counterelectrodes). In this embodiment, the sample chamber and measurement zoneare effectively the same and have the same volume. In other embodiments,the measurement zone has, for example, 80% or 90% of the volume of thesample chamber. It will be understood that similar sensors could be madeusing one counter electrode or three or more counter electrodes. It willalso be understood that multiple working electrodes may also be providedon the sensor.

One example of a method for making the thin film sensors is describedwith respect to the sensor arrangement displayed in FIGS. 18A to 18C andcan be used to make a variety of other sensor arrangements, includingthose described before. A substrate, such as a plastic substrate, isprovided. The substrate can be an individual sheet or a continuous rollon a web. This substrate can be used to make a single sensor or to makemultiple sensors. The multiple sensors can be formed on a substrate 1000as working electrodes 1010 and counter electrode(s) 1020. In someembodiments, the substrate can be scored and folded to bring the workingelectrodes 1010 and counter electrodes 1020 together to form the sensor.In some embodiments, as illustrated in FIG. 31A, the individual workingelectrodes 1010 (and, in a separate section, the counter electrode(s)1020) can be formed next to each other on the substrate 1000, to reducewaste material, as illustrated in FIG. 31A. In other embodiments, theindividual working electrodes 1010 (and, in a separate section, thecounter electrode(s) 1020) can be spaced apart, as illustrated in FIG.31B. The remainder of the process is described for the manufacture ofmultiple sensors, but can be readily modified to form individualsensors.

Carbon or other electrode material (e.g., metal, such as gold orplatinum) is formed on the substrate to provide a working electrode foreach sensor. The carbon or other electrode material can be deposited bya variety of methods including printing a carbon or metal ink, vapordeposition, and other methods.

Optionally, a non-conductive material, such as a non-conductive ink, canbe formed adjacent the working electrode to provide a planar surfacealong the path of travel of the sample fluid. The non-conductivematerial is suitable for creating a smooth surface to facilitate fillingby capillary action and/or for reducing the likelihood that air bubbleswill become entrapped near the working electrode. This non-conductivematerial can be colored or colorless and may be formed on the substrateby printing or other techniques. The non-conductive material may bedeposited prior to or subsequent to the formation of the workingelectrode.

The counter electrode or counter electrodes are formed on the substrate.The counter electrode(s) are formed by depositing carbon or otherelectrode material onto the substrate. In one embodiment, the materialof the counter electrode(s) is a Ag/AgCl ink. The material of thecounter electrode(s) may be deposited by a variety of methods includingprinting or vapor deposition. In some embodiments, the counterelectrodes are formed using different materials and/or one electrode isa counter or counter/reference electrode and the other electrode is areference or counter/reference electrode. In one embodiment, the workingelectrodes are formed on a first half of a polymer sheet or web and thecounter electrodes are formed on a second half of the polymer sheet orweb so that the sheet or web can be folded to superimpose the workingand counter electrodes in a facing arrangement.

A second non-conductive material may be deposited adjacent and/orbetween the counter electrode(s) to provide a planar surface along thepath of travel of the sample fluid. This may be particularly desirablein the region between the counter electrodes that will be part of thesample chamber to planarize the surface of the sample chamber. Thenon-conductive material is suitable for creating a smooth surface tofacilitate filling by capillary action and/or for reducing thelikelihood that air bubbles will become entrapped between or near thecounter electrode(s). This non-conductive material can be colored orcolorless and may be formed on the by printing or other techniques. Thenon-conductive material may be deposited prior to or subsequent to theformation of the counter electrode(s).

An adhesive spacer is formed over at least one of the substrate/workingelectrode and substrate/counter electrode(s). The adhesive spacer may bea single layer of adhesive or a double-sided adhesive tape (e.g., apolymer carrier film with adhesive disposed on opposing surfaces). Toform the channel, the spacer, optionally provided with one or morerelease liners, may be cut (e.g., die-cut) to remove the portion of theadhesive corresponding to the channel prior to disposing the spacer onthe substrate. Alternatively, the adhesive may be printed or otherwisedisposed on the substrate according to a pattern which defines thechannel region. The thickness of the spacer typically determines thespacing between the working and counter electrodes. When the uniformityof this spacing among sensors is necessary (e.g., for coulometricmeasurements), uniformity in the thickness of the spacer is important.Preferably, the thickness does not vary more than ±5% over theindividual sensor and/or among individual sensors in a batch.

The non-leachable or diffusible redox mediator and/or second electrontransfer agent are disposed onto the substrate in at least the samplechamber region. If either or both of these components is non-leachable,that component or components must be disposed on the working electrode.If either or both of these components is diffusible, that component orcomponents can be disposed on any surface of the substrate in thechannel region. The redox mediator and/or second electrode transferagent can be disposed independently or together on the substrate priorto or after disposition of the spacer. The redox mediator and/or secondelectrode transfer agent may be disposed by a variety of methodsincluding, for example, screen printing, ink jet printing, spraying,painting, striping along a row or column of aligned and/or adjacentelectrodes, and the like. Other components may be deposited separatelyor with the redox mediator and/or second electrode transfer agentincluding, for example, surfactants, polymers, polymer films,preservatives, binders, buffers, and cross-linkers.

After disposing the spacer, redox mediator, and second electron transferagent, the substrate can be folded to form the sensor. The faces of thesubstrate are joined by the adhesive of the spacer. After bringing thefaces together, the sensor can be cut out using a variety of methodsincluding, for example, die cutting, slitting, or otherwise cutting awaythe excess substrate material and separating the individual sensors. Insome embodiments, a combination of methods may be used. For example,some features may be die cut, while the remainder of the sensor is cutby slitting. As another alternative, the sensor components (e.g., thecomponents illustrated in FIGS. 18A and 18C) may first be cut out of thesubstrates and then brought together to form the sensor by adhesivelyjoining the two components using the spacer adhesive.

The embodiment of a sensor illustrated in FIGS. 18A to 18C is an exampleof a tip-fill sensor. An alternative sensor construction is illustratedin FIGS. 19A to 19C. This is a side-fill sensor. FIG. 19A illustrates afirst substrate 520 with a working electrode 522. FIG. 19B illustrates aspacer 524 defining a channel 526. FIG. 19C (inverted with respect toFIGS. 19A and 19B to illustrate the electrodes) illustrates a secondsubstrate 528 with three counter (or counter/reference) electrodes 530,532, 534.

This sensor can be manufactured as described above. The symmetricdisposition of the counter electrodes allow the sensor to be filled fromeither the left or right side for convenience of left-handed andright-handed people. It will be understood, however, that similar sensorarrangements can be formed using one, two, or four or more counterelectrode(s) and/or two or more working electrodes. The scallopedregions 536, 538 may be formed, for example, by die cutting and may, atleast in some instances, be precisely controlled to provide areproducible channel length. As an alternative arrangement, the sides ofthe sensor may be straight to allow the sensor to be cut out from theremainder of the substrate and/or from other sensors by slitting thesubstrate in parallel directions using, for example, a gang arbor bladesystem. As illustrated in FIGS. 19A, 19B, and 19C, the edges of thesensor can define edges of the sample chamber and/or measurement zone.By accurately controlling the distance between cuts, variability insample chamber volume can often be reduced. In some instances, thesecuts are preferably parallel to each other, as parallel cuts may be theeasiest to make reproducibly.

FIGS. 20A, 20B, and 20C illustrate another example of a side-fillingsensor arrangement. FIG. 20A illustrates a first substrate 540 with aworking electrode 542. FIG. 20B illustrates a spacer 544 defining achannel 546. FIG. 20C (inverted with respect to FIGS. 20A and 20B)illustrates a second substrate 548 with three counter (orcounter/reference) electrodes 550, 552, 554.

FIGS. 21A, 21B, and 21C illustrate another example of a tip-fillingsensor arrangement. FIG. 21A illustrates a first substrate 560 with aworking electrode 562. FIG. 21B illustrates a spacer 564 defining achannel 566. FIG. 21C (inverted with respect to FIGS. 21A and 21B)illustrates a second thin film substrate 568 with two counter (orcounter/reference) electrodes 570, 572. A vent hole 574 (indicated as ashaded region in FIG. 21C) is provided through the second substrate. Inthe illustrated embodiment, this vent hole 574 is made through only thesubstrate 568 that carries the counter electrode(s) and, optionally, thespacer 564. In this embodiment, the vent hole may be formed by, forexample, die cutting a portion of the substrate. This die cut may removea portion of at least one counter electrode, but a sufficient amount ofthe counter electrode should remain for contact with the sample in thechannel and for electrical connection to a contact at the other end ofthe sensor. In another embodiment, the vent hole 574 may be made throughall of the layers or through the first substrate and not the secondsubstrate.

Another embodiment is illustrated in FIGS. 22A, 22B, and 22C, with adifferent shape. This sensor includes a first substrate 579 with atleast one working electrode 580, as illustrated in FIG. 22A. The sensoralso includes a spacer 581 with a channel 582 formed in the spacer 581,as shown in FIG. 22B. The sensor further includes a second substrate 583with two counter electrodes 584, 585, as shown in FIG. 22C (invertedwith respect to FIGS. 22A and 22B). A venting aperture 586 is cuttypically through all of the layers and extends from a side of thesensor. In some embodiments, the venting aperture and the front portion587 of the sensor are simultaneously cut with a reproducible distancebetween the venting aperture and the front portion 587 of the sensor toprovide a reproducible length for the channel 582 and the workingelectrode 580. FIGS. 22A, 22B, and 22C also illustrate another featurethat can be used with any sensor arrangement. An indentation 588 may beformed at the filling opening of the channel 582 to facilitate thedrawing of fluid into the sensor. In this configuration, the fluid isnot provided with a flat face, but rather an indented face that may aidin wicking or capillary filling of the channel (i.e., sample chamber).This configuration may also reduce the likelihood that the user of thesensor will block the channel during collection of the sample. A flatfaced sensor might be blocked by pressing the tip of the sensor edgewiseagainst the skin.

FIGS. 23A, 23B, and 23C illustrate another example of a side-fillingsensor arrangement. FIG. 23A illustrates a first substrate 640 with aworking electrode 642. FIG. 23B illustrates a spacer 644 defining achannel 646. FIG. 23C (inverted with respect to FIGS. 23A and 23B)illustrates a second substrate 648 with three counter (orcounter/reference) electrodes 650, 652, 654. This sensor can be formedby making straight cuts of the substrates. The sensors can be madeadjacent to one another, as illustrated in FIG. 31A, which may produceless waste material. The length of the channel 646 is typically definedby the two parallel cuts along the sides 656, 658 of the sensors.Another optional processing advantage, particularly if the sensor areformed adjacent to each other, is that the redox mediator and/or secondelectron transfer agent can be disposed in the channel by striping acontinuous stream of these components along a row or column of adjacentsensors. This may result in better efficiency and less waste of theredox mediator and/or second electron transfer agent, as compared toother techniques, such as individually placing these components withinthe individual channels.

FIGS. 24A, 24B, and 24C illustrate another sensor configuration. Thissensor includes a first substrate 600 with at least one workingelectrode 602, as illustrated in FIG. 24A. The sensor also includes aspacer 604 with a channel 606 formed in the spacer 604, as shown in FIG.24B. The sensor further includes a second substrate 608 with two counterelectrodes 610, 612, as shown in FIG. 24C (inverted with respect toFIGS. 24A and 24B). The sensor may also include, for example, anindicator, such as a slot 614 or an extension 616 from the body of thesensor that indicates to the user which side should be placed adjacentto the sample. This may be particularly important where the sensorreading is only correct when sample enters from a particular side.

FIG. 24B also illustrates another optional feature that may be used inany of the sensor configurations. In this illustration, the samplechamber 606 is not formed using straight lines, but there is an expandedregion 618 within the sample chamber. This permits larger samplechambers without forming larger openings. This expanded region can beformed as any shape including circular, square, rectangular, and otherregular and irregular shapes.

FIG. 25 is an example of an assembled sensor illustrating anotheralternative sensor arrangement for a side-fill sensor 620. This sensorincludes extensions 622 from the sensor body 624 to indicate to a userwhere the openings for the sample chamber 626 are provided.

One optional feature is illustrated in FIG. 32 which is an edge-on viewof the sensor from the inside of the meter. FIG. 32 illustrates a firstsubstrate 1120 and a second substrate 1130 that extend into the meterfrom the remainder of the sensor 1100 (i.e., portion 1140 is recessedwith respect to substrates 1120 and 1130 in FIG. 32). Examples of thisconfiguration are illustrated in FIGS. 18A-18C and 24A-24C. Typically,the sensor 1100 is coupled to a meter 1110 that includes contact pads(not shown) that contact the contact regions (e.g., regions 503, 511,and 513 in FIGS. 18A and 18C) of the electrodes of the sensor 1100. Theend of the sensor 1100 which contains the contact regions can be slidinto the meter 1110. It is typically important that the contact pads ofthe meter 1110 make contact with the correct contact regions of thesensor so that the working electrode and counter electrode(s) arecorrectly coupled to the meter. In some instances, the sensor isconfigured so that the contact region for the working electrode on thefirst substrate 1120 has a different width, w1, than width, w2, for thecontact region of the second substrate 1130 carrying the counterelectrode(s). Examples of electrode configurations with this structureare provided in FIGS. 18A-18C and 24A-24C. To ensure proper insertion ofthe sensor 1100 into the meter 1110, the meter 1110 may include a raisedarea 1140 that prevents or hinders the insertion of the sensor in animproper direction. For example, the width, w2, of the contact region ofthe second substrate 1130 may be wider than the width, w1, of thecontact region of the first substrate 1120, as illustrated in FIG. 32.In this instance, the raised area 1140 is positioned to allow sensor1100 to be slid into the meter so that the first substrate 1120 is nextto the surface 1150 from which the raised area 1140 protrudes, but wouldprevent or hinder having the second substrate 1130 next to the surface1150 from which the raised area 1140 protrudes. Objects other than araised area can also be used to guide the user in correct introductionof the sensor into the meter.

Integrated Sample Acquisition and Analyte Measurement Device

Many approaches are known in the art for acquiring and/or transporting asmall sample from the body to a sensor. These include, for example, U.S.Pat. Nos. 5,746,217; 5,820,570; 5,857,983; and 5,879,311, incorporatedherein by reference. Any of these sample acquisition and/or transportingmethods may be employed with the sensor of the current invention.

In a preferred embodiment of the invention, an analyte measurementdevice 52 constructed according to the principles of the presentinvention includes a sensor 20, as described hereinabove, combined witha sample acquisition apparatus 50 to provide an integrated sampling andmeasurement device. The sample acquisition apparatus 50 illustrated inFIG. 6, includes, for example, a skin piercing member 54, such as alancet, attached to a resilient deflectable strip 56 (or other similardevice, such as a spring) which may be pushed to inject the lancet intoa patient's skin to cause blood flow.

The resilient strip 56 is then released and the skin piercing member 54retracts. Blood flowing from the area of skin pierced by member 54 canthen be transported, for example, by the wicking action of sorbentmaterial 34, into sensor 20 for analysis of the analyte. The analytemeasurement device 52 may then be placed in a reader, not shown, whichconnects a coulometer or other electrochemical analysis equipment to theelectrode tabs 23, 25 to determine the concentration of the analyte byelectroanalytical means. Preferably, the analyte measurement device isenclosed within the reader when connected to the coulometer or otherelectrochemical analysis equipment.

In a preferred embodiment, the integrated sample acquisition and analytemeasurement device comprises a lancing instrument that holds a lancetand measurement strip. The lancing instrument preferably requires activecocking. By requiring the user to cock the device prior to use, the riskof inadvertently triggering the lancet is minimized.

Preferably, the lancing instrument is automatically triggered when thelancing instrument is pressed firmly against the skin with an adequateamount of pressure. As is already known in the art, a larger sample ofbody fluid such as blood or interstitial fluid is expressed whenpressure is applied around a site where a hole has been created theskin. For example, see the above-mentioned U.S. patents to Integ andAmira as well as the tip design of the lancing instruments sold byBecton Dickenson. All of these lancing devices have a protruding ringthat surrounds the lancing site to create pressure that forces sampleout of the wound. However, all of these devices require the user toapply adequate pressure to the wound site to express the sample, and allof the lancing instruments are triggered by a button push by the user.Design of an appropriate pressure trigger is well-known to one skilledin the art.

Preferably, the lancing instrument will also permit the user to adjustthe depth of penetration of the lancet into the skin. Such devices arealready commercially available from companies such as BoehringerMannheim and Palco. This feature allows users to adjust the lancingdevice for differences in skin thickness, skin durability, and painsensitivity across different sites on the body and across differentusers.

In a more preferred embodiment, the lancing instrument and the testreader are integrated into a single device. To operate the device theuser need only insert a disposable cartridge containing a measurementstrip and lancing device into the integrated device, cock the lancinginstrument, press it against the skin to activate it, and read theresult of the measurement. Such an integrated lancing instrument andtest reader simplifies the testing procedure for the user and minimizesthe handling of body fluids.

FIG. 26 illustrates another example of an integrated sample acquisitionand sensor device 700. The integrated sample acquisition and sensordevice 700 includes a housing 702, a skin piercing member (e.g., alancet) 704, a piercing/collecting aperture 706, an optionally removablesensor 708, a sensor guide 710, and a retraction mechanism 714 for theskin piercing member. This device 700 can be designed for reuse (e.g.,by making the skin piercing member 704 and sensor 708 removable) or forsingle use.

The housing 702 may be formed of a variety of materials including metaland plastic. The housing 702 may include a hinge 716 or otherconfiguration (e.g., adhesive or interlocking parts) for holdingportions of the housing together.

The piercing/collecting aperture 706 is provided in the housing 702 toallow the skin piercing member 704 to extend through the aperture 706and pierce the skin of a user, thereby causing blood (or other bodyfluid) flow. The sensor 708 also extends to the edge or out of theaperture 706 to collect the blood (or other body fluid) through anopening (not shown) in the tip of the sensor. This may allow the user topierce the skin and collect the fluid sample without moving the device700. Alternatively, separate apertures may be provided for the skinpiercing member 704 and sensor 708. The sensor guide may be formed inthe housing 702 or added to the housing to guide the sensor 708 intoplace if the sensor is inserted into and through the housing and/or tosupport the sensor within the housing and during sample collection.

The skin piercing member 704 may include an actuator (not shown) thatincludes a mechanism that allows for cocking and releasing the skinpiercing member 704 or the skin piercing member may be actuatedexternally. For example, a sensor reader (not shown) or other device maybe coupled to the sample acquisition and sensor device, the sensorreader or other device including a mechanism that cocks and/or releasesthe skin piercing member 704.

The retraction mechanism 714 of the device 700 may be, for example, aspring or resilient metal strip that retracts the skin piercing member704 back into the housing after piercing the skin of the user. This mayallow for unobstructed collection of the sample and/or prevent furtherpiercing of the skin of the user or others to reduce or preventcontamination or infection caused by transfer of body fluids or otherharmful agents. Alternatively, retraction of the skin piercing membermay be accomplished using an external device or apparatus.

One example of operation includes cocking the skin piercing member 704and then releasing the skin piercing member 704 so that it extends outof the housing 702 through the piercing/collecting aperture 706 andpierces the skin of the user. The skin piercing element 704 optionallypushes the sensor out of the way while extending out of the housing. Theskin piercing element 704 is retracted back within the housing 702 usingthe retraction mechanism 714. Upon retraction of the skin piercingelement, the sensor collects a sample fluid from the pierced skinthrough an opening in the sensor 708.

If a sensor reader is used, the sensor reader may also be configured tocouple with a contact end of the sensor. The sensor reader may include apotentiostat or other component to provide a potential and/or currentfor the electrodes of the sensor. The sensor reader may also include aprocessor (e.g., a microprocessor or hardware) for determining analyteconcentration from the sensor signals. The sensor reader may include adisplay or a port for coupling a display to the sensor. The display maydisplay the sensor signals and/or results determined from the sensorsignals including, for example, analyte concentration, rate of change ofanalyte concentration, and/or the exceeding of a threshold analyteconcentration (indicating, for example, hypo- or hyperglycemia). Thissensor reader may be used in conjunction with the integrated sampleacquisition and sensor device or the sensor reader may be used with thesensor alone, the contacts of the sensor making connection with contactsin the sensor reader.

Operation of the Sensor

An electrochemical sensor of the invention may be operated with orwithout applying a potential. In one embodiment, the electrochemicalreaction occurs spontaneously and a potential need not be appliedbetween the working and counter electrodes.

In another embodiment, a potential is applied between the working andcounter electrodes. Yet the potential does not need to remain constant.The magnitude of the required potential is dependent on the redoxmediator. The potential at which the electrode poises itself, or whereit is poised by applying an external bias, and where the analyte iselectrolyzed is typically such that the electrochemical reaction isdriven to or near completion, but it is, preferably, not oxidizingenough to result in significant electrochemical reaction ofinterferents, such as urate, ascorbate, and acetaminophen, that mayaffect the signal measured. For non-leachable redox mediators, thepotential is typically between about −350 mV and about +400 mV versusthe standard calomel electrode (SCE). Preferably, the potential of theredox mediator is more negative than +100 mV, more preferably thepotential is more negative than 0 mV, and most preferably the potentialis about −150 mV versus SCE.

When an external potential is applied, it may be applied either beforeor after the sample has been placed in the sample chamber. If themeasurement zone comprises only a portion of the sample chamber then thepotential is preferably applied after the sample has come to rest in thesample chamber to prevent electrolysis of sample passing through themeasurement zone as the sample chamber is filling. Alternatively, in thecase where the measurement zone comprises most or all of the samplechamber, the potential, optionally, may be applied before or during thefilling of the sample chamber without affecting the accuracy of theassay. When the potential is applied and the sample is in themeasurement zone, an electrical current will flow between the workingelectrode and the counter electrode. The current is a result, at leastin part, of the electrolysis of the analyte in the sample. Thiselectrochemical reaction occurs via the redox mediator and the optionalsecond electron transfer agent. For many biomolecules, B, the process isdescribed by the following reaction equations:

Biochemical B is oxidized to C by redox mediator species A in thepresence of an appropriate enzyme. Then the redox mediator A is oxidizedat the electrode. Electrons are collected by the electrode and theresulting current is measured. The measured current may also include abackground current resulting in a measured background charge, due, atleast in part, to the shuttling of a diffusible redox mediator betweenthe working electrode and the counter electrode. This background currentcan be minimized or accounted for, as described above.

As an example, one sensor of the present invention is based on thereaction of a glucose molecule with two [Os(dmo-phen)₂(NMI)Cl]²⁺cations, where dmo-phen is 4,8-dimethoxy phenanthroline and NMI isN-methyl-imidazole, in the presence of glucose oxidase to produce two[Os(dmo-phen)₂(NMI)Cl]⁺ cations, two protons, and an oxidation productof glucose, for example, gluconolactone or another ketone. The amount ofglucose present is assayed by electrooxidizing the[Os(dmo-phen)₂(NMI)Cl]⁺ cations to [Os(dmo-phen)₂(NMI)Cl]²⁺ cations andmeasuring the total charge passed.

Those skilled in the art will recognize that there are many differentreactions that will provide the same result; namely the electrolysis ofan analyte through a reaction pathway incorporating a redox mediator.Equations (1) and (2) are a non-limiting example of such a reaction.

Coulometry

In a preferred embodiment of the invention, coulometry is used todetermine the concentration of the analyte. This measurement techniqueutilizes current measurements obtained at intervals over the course ofthe assay, to determine analyte concentration. These currentmeasurements are integrated over time to obtain the amount of charge, Q,passed to or from the electrode. Q is then used to calculate theconcentration of the analyte (C_(A)) by the following equation (when theredox mediator is non-leachable):C _(A) =Q/nFV  (3a)where n is the number of electron equivalents required to electrolyzethe analyte, F is Faraday's constant (approximately 96,500 coulombs perequivalent), and V is the volume of sample in the measurement zone. Whenusing a diffusible mediator, the concentration of the analyte can beobtained from the following equation:C _(A)+(Q _(tot) −Q _(back))/nFV  (3b)where Q_(tot) is the total charge transferred during the measurement andQ_(back) is the amount of charge transferred that was not due to theanalyte, e.g., charge transferred by the shuttling of the diffusiblemediator between the working electrode and the counter electrode. In atleast some instances, the sensor is constructed so that the backgroundto charge is at most 5 times the size of the charge generated byelectrolysis of an amount of analyte. Preferably, the background signalis at most 200%, 100%, 50%, 25%, 10%, or 5% of the charge generated byelectrolysis of the analyte.

One example of a method for determining the ratio of background signalto signal generated by electrolysis of the analyte is described asfollows for the facing electrode pairs. If the shuttling of the redoxmediator is not disabled by the applied potential, the charge thatresults from the shuttling of the redox mediator may be represented bythe following formula:Q _(back)=(AFD _(M) C _(M) /d)(tn _(M))where A is the area of the working electrode; F is Faraday's constant(96,500 coulombs/equivalent); D_(M) is the effective diffusioncoefficient of the redox mediator; C_(M) is the concentration of theredox mediator in the measurement zone; d is the distance separatingfacing electrodes; t is the amount of time for the measurement; andn_(M) is the number of electrons gained or lost by the redox mediator.

Additionally, the charge of the analyte, for example, glucose, when theanalyte is electrooxidized to about 90% completion in the measurementperiod may be represented by the following formula:Q _(G) =Ad(0.90)C _(G) n _(G) Fwhere A is the area of the working electrode; d is the distanceseparating facing electrodes; C_(G) is the concentration of glucose; nis the number of electrons needed to electrolyze the analyte (e.g., 2electrons per glucose molecule); and F is Faraday's constant. When C_(G)is 5 mM (or 5×10⁻⁶ moles/cm³), t is 60 seconds, n_(G) is 2, and n_(M) is1, the ratio of charge from the redox mediator to the charge fromelectrooxidation of the analyte may be represented by the followingformula:Q _(Back) /Q _(G)=(D _(M) C _(M) /d ²)(tn _(M)/(0.9 n _(G) C _(G)))=(D_(M) C _(M) /d ²)×(6.7×10⁶)For example, if the ratio of Q_(Back)/Q_(G) is 5, then (D_(M) C_(M))/d²is 7.5×10⁻⁷ moles/(cm³ sec). Also for example, if the ratio ofQ_(Back)/Q_(G) is 1, then (D_(M) C_(M))/d² is 1.5×10⁻⁷ moles/(cm³ sec).Still another example, if the ratio is 0.1, then (D_(M) C_(M))/d² is1.5×10⁻⁸ moles/(cm³ sec). Thus, depending on the ratio desired, a sensormay be configured to have the desired ratio by choosing D_(M), C_(M),and d accordingly. For example, the concentration of the redox mediatormay be reduced (i.e., C_(M) may be reduced). Alternatively, oradditionally, the diffusion of the redox mediator may be reduced by, forexample, having a barrier to the flow of the diffusible mediator to thecounter electrode (i.e., reduce the effective diffusion coefficient ofthe redox mediator—D_(M)). Other sensor configurations are also suitablefor controlling the ratio of background signal to signal generated bythe analyte and will be described below.

The background charge, Q_(back), can be accounted for in a variety ofways. Q_(back) can be made small, for example, by using only limitedamounts of diffusible redox mediator; by providing a membrane over thecounter electrode that limits diffusion of the redox mediator to thecounter electrode; or by having a relatively small potential differencebetween the working electrode and the counter electrode. Other examplesof sensor configurations and methods suitable for reducing Q_(back)include those already described such as sensors having a redox mediatorreaction rate at the working electrode that is significantly faster thanthat at the counter electrode; immobilizing the redox mediator on theworking electrode; having the redox mediator become immobilized on thecounter or counter/reference electrode upon its reaction at the counteror counter/reference electrode; or slowing the diffusion of the redoxmediator.

Alternatively, the sensor may be calibrated individually or by batch todetermine a calibration curve or a value for Q_(back). Another option isto include a second electrode pair that is missing an item necessary forelectrolysis of the analyte, such as, for example, the second electrontransfer agent, so that the entire signal from this second electrodepair corresponds to Q_(back).

For coulometric measurements, at least 20% of the analyte iselectrolyzed. Preferably at least 50%, more preferably at least 80%, andeven more preferably at least 90% of the analyte is electrolyzed. In oneembodiment of the invention, the analyte is completely or nearlycompletely electrolyzed. The charge can then be calculated from currentmeasurements made during the electrochemical reaction, and theconcentration of the analyte is determined using equation (3a) or (3b).The completion of the electrochemical reaction is typically signaledwhen the current reaches a steady-state value. This indicates that allor nearly all of the analyte has been electrolyzed. For this type ofmeasurement, at least 90% of the analyte is typically electrolyzed,preferably, at least 95% of the analyte is electrolyzed and, morepreferably, at least 99% of the analyte is electrolyzed.

For coulometry, it is typically desirable that the analyte beelectrolyzed quickly. The speed of the electrochemical reaction dependson several factors, including the potential that is applied between theelectrodes and the kinetics of reactions (1) and (2). (Other significantfactors include the size of the measurement zone and the presence ofsorbent in the measurement zone.) In general, the larger the potential,the larger the current through the cell (up to a transport limitedmaximum) and therefore, the faster the reaction will typically occur.However, if the potential is too large, other electrochemical reactionsmay introduce significant error in the measurement. Typically, thepotential between the electrodes as well as the specific redox mediatorand optional second electron transfer agent are chosen so that theanalyte will be almost completely electrolyzed in less than 5 minutes,based on the expected concentration of the analyte in the sample.Preferably, the analyte will be almost completely electrolyzed withinabout 2 minutes and, more preferably, within about 1 minute.

In another embodiment of the invention, the analyte is only partiallyelectrolyzed. The current is measured during the partial reaction andthen extrapolated using mathematical techniques known to those skilledin the art to determine the current curve for the complete or nearlycomplete electrolysis of the analyte. Integration of this curve yieldsthe amount of charge that would be passed if the analyte were completelyor nearly completely electrolyzed and, using equation (3a) or (3b), theconcentration of the analyte is calculated.

Although coulometry has the disadvantage of requiring the volume of themeasured sample be known, coulometry is a preferred technique for theanalysis of the small sample because it has the advantages of, forexample, no temperature dependence for the measurement, no enzymeactivity dependence for the measurement, no redox-mediator activitydependence for the measurement, and no error in the measurement fromdepletion of analyte in the sample. As already described above,coulometry is a method for determining the amount of charge passed orprojected to pass during complete or nearly complete electrolysis of theanalyte. One coulometric technique involves electrolyzing the analyte ona working electrode and measuring the resulting current between theworking electrode and a counter electrode at two or more times duringthe electrolysis. The electrolysis is complete when the current reachesa steady state. The charge used to electrolyze the sample is thencalculated by integrating the measured currents over time and accountingfor any background signal. Because the charge is directly related to theamount of analyte in the sample there is no temperature dependence ofthe measurement. In addition, the activity of the enzyme does not affectthe value of the measurement, but only the time required to obtain themeasurement (i.e., less active enzyme requires a longer time to achievecomplete electrolysis of the sample) so that decay of the enzyme overtime will not render the analyte concentration determination inaccurate.And finally, the depletion of the analyte in the sample by electrolysisis not a source of error, but rather the objective of the technique.(However, the analyte need not be completely electrolyzed if theelectrolysis curve is extrapolated from the partial electrolysis curvebased on well-known electrochemical principles.)

Non-Coulometric Assays

Although coulometric assays are useful, those skilled in the art willrecognize that a sensor of the invention may also utilizepotentiometric, amperometric, voltammetric, and other electrochemicaltechniques to determine the concentration of an analyte in a sample. Themeasurements obtained by these non-coulometric methods may not betemperature independent as are coulometric measurements.

In addition, the measurements obtained by these non-coulometricelectrochemical techniques may be sensitive to the amount of activeenzyme provided in the sensor. If the enzyme deactivates or decays overtime, the resulting measurements may be affected. This may limit theshelf life of such sensors unless the enzyme is very stable.

Finally, the measurements obtained by non-coulometric electrochemicaltechniques, such as steady-state amperometry, may be negatively affectedif a substantial portion of the analyte and/or redox mediator iselectrolyzed during the measurement period. An accurate steady-statemeasurement may not be obtainable unless there is sufficient analyteand/or redox mediator so that only a relatively small portion of theanalyte and/or redox mediator is electrolyzed during the measurementprocess. This may be challenging in a sample size of no more than 1 μl.

It may be desirable in some instances to utilize non-coulometric assays,such as amperometric or potentiometric measurement techniques. Forexample, coulometry requires that the volume of the measured sample beknown. And, the volume of the sample in the measurement zone of a smallvolume sensor (i.e., no more than one microliter) may be difficult toaccurately reproduce if the manufacturing tolerances of one or moredimensions of the measurement zone have significant variances.

As described for coulometric measurements, the background signalresulting from the shuttling of the redox mediator between theelectrodes can be a source of measurement error in amperometric orpotentiometric assays of samples of no more than 1 μL in thin layerelectrochemical cells. In general, it is desirable that the mediatordoes not shuttle between a pair of electrodes more than ten times in theperiod of the measurement, preferably not more than once, and morepreferably not more than 0.1 times, on average. To decrease errorarising from background signal, methods and sensor configurationssimilar to, and in some cases identical to, those used for coulometricmeasurements may be used. Examples include all of the methods andstructures described above, such as performing the electrochemical assayat relatively low applied potential, electrooxidizing the analyte atnegative applied potentials or electroreducing the analyte at positiveapplied potentials, using a counter electrode at which the redoxmediator reacts relatively slowly (particularly as compared to thereaction of the redox mediator at the working electrode), and/or using aredox mediator that undergoes an irreversible reaction at the counterelectrode. Other examples are discussed below.

As described for coulometric measurements, it is preferred that thesensor be designed and operated so that the background signal is at mostfive times the size of the signal generated by electrolysis of theanalyte. Preferably, the background signal is at most 200%, 100%, 50%,25%, 10%, or 5% of the signal generated by electrolysis of an amount ofanalyte. The amount of analyte against which the background signal iscompared is described above in the section entitled “Background Signal.”In the case of amperometry, the signal generated by electrolysis of anamount of analyte is the current at the time or times at which themeasurement is taken. In the case of potentiometry, the signal generatedby electrolysis of an amount of analyte is the potential at the time ortimes at which the measurement is taken.

Under a given set of operating conditions, for example, temperature,cell geometry, and electrode size, the magnitude of the backgroundcurrent, I_(back), is given by the following expression:i _(back) =KC _(M) D _(M) /dwhere: K is a proportionality constant; C_(M) is the concentration ofthe mediator in the measurement zone; D_(m) is the effective diffusioncoefficient of the mediator in the measurement zone under normaloperating conditions; and d is the distance between the electrodes.

It is desirable to reduce background current for non-coulometric assays.The sensor configurations and methods described above are generallyuseful and include, for example, using low concentrations of the redoxmediator and/or the second electron transfer agent (e.g., enzyme)relative to the concentration of the analyte and/or using a large redoxmediator having a relatively low effective diffusion constant. Otheruseful methods described above include methods for reducing thediffusion of the redox mediator by, for example, having a barrier (e.g.,a charged or polar barrier) to the flow of the diffusible mediator orusing a redox mediator having a relatively low effective diffusionconstant.

In some instances, the effective diffusion coefficient is no more thanabout 1×10⁻⁶ cm²/sec, no more than about 1×10⁻⁷ cm²/sec, or no more thanabout 1×10⁻⁸ cm²/sec. Moreover, in some cases, the product of C_(M)D_(M)(the concentration of redox mediator times the effective diffusioncoefficient) is no more than about 1×10⁻¹² moles/cm·sec, no more thanabout 1×10⁻¹³ moles/cm·sec, or no more than about 1×10⁻¹⁴ moles/cm·sec.

The following provides a specific example for the case of anamperometric measurement of glucose carried out for 60 seconds duringwhich time 10% of the glucose is electrolyzed in a 1 microliter cellwith facing electrodes separated by a distance of d=0.01 cm. If themeasurement was carried out under the following conditions: a glucoseconcentration of C_(G)=5 mM (or 5×10⁻⁶ moles/cm³), an area of A=0.1 cm²,a number of electrons from the redox mediator of n_(M)=1, and a numberof electrons from glucose n_(G)=2, then the background current generatedby the redox mediator and by the glucose is determined as follows.

$\begin{matrix}\begin{matrix}{i_{back} = {{AFn}_{M}D_{M}{C_{M}/d}}} \\{= {(0.1)\left( {96\text{,}500} \right)(1)D_{M}{C_{M}/(0.01)}}} \\{= {9.65 \times 10^{5}C_{M}D_{M}}}\end{matrix} & \; \\\begin{matrix}{i_{G} = {n_{G}{{Ad}(0.1)}{{FC}_{G}/t}}} \\{= {(2)(0.01)(0.1)\left( {96\text{,}500} \right){\left( {5 \times 10^{- 6}} \right)/60}}} \\{= {1.61\mspace{14mu}{\mu amps}}}\end{matrix} & \;\end{matrix}$

Thus if i_(back)/i_(G)=5, the value of C_(M)D_(M) equal 8.34×10⁻¹²moles/cm² sec. As another example, if i_(back)/i_(G)=0.5, the value ofC_(M)D_(M) equal 8.34×10⁻¹³ moles/cm² sec. Additionally ifi_(back)/i_(G)=0.05, the value of C_(M)D_(M) equal 8.34×10⁻¹⁴ moles/cm²sec.

In some amperometric or potentiometric embodiments, the redox mediatorcirculation is decreased by separating the working electrode from thecounter or counter/reference electrode such that the distance throughwhich the redox mediator would diffuse during the measurement period isno greater than, for example, the distance between the electrodes. Aredox mediator can diffuse a distance equal to (D_(m)t)^(1/2), whereD_(m) is the effective diffusion coefficient for the medium between theelectrodes and t is time. For a measurement time period of 30 secondsand a redox mediator with effective diffusion coefficient between 10⁻⁵and 10⁻⁶ cm²/second, the electrodes should be separated by at least 100μm, preferably at least 200 μm, and even more preferably at least 400μm.

One method of separating the working and counter electrodes is to use athicker spacer between the electrodes. One alternative method isillustrated in FIG. 27. In this embodiment, the working electrode 740 isdisposed on a first substrate 742 and the counter electrode 744 isdisposed on a second substrate 746 (alternatively, the electrodes may bedisposed on the same substrate). The working electrode 742 and thecounter electrode 744 are offset so that the effective distance, d,between the two electrodes is greater than the thickness, w, of thespacer layer 748. In one embodiment, the distance between theelectrodes, d, is selected to be in the range of 25 to 1000 μm, 50 to500 μM, or 100 to 250 μm.

Additionally or alternatively, in the case of steady-state amperometryand potentiometry, background signal may be controlled by limiting therate of electrolysis such that the rate is slow enough to prevent theanalyte concentration from decreasing by more than about 20%, 10%, or 5%or less, during a measurement period, e.g., 30 second, 1 minute, 5minutes, or 10 minutes. In some instances, to control the rate ofelectrolysis the concentration or activity of the second electrontransfer agent may be reduced and/or the working electrode area may bereduced.

For example, the second electron transfer agent can be an enzyme and theenzyme activity can be a limiting factor for the electrolysis rate. If,for example, the analyte concentration is 5 mM glucose (i.e., 5×10⁻⁹moles of glucose in 1 μl) and no more than 10% of the glucose (5×10⁻¹⁰moles) is to be electrooxidized during a 30-second measurement period,the current should not exceed 3.3×10⁻⁶ amperes for 1 μL. One unit of anenzyme is that amount of the enzyme which catalyzes electrolysis of 1μmole of its substrate in 60 seconds at pH of 7.4 at 37° C. in HEPESbuffer. Accordingly, for glucose, a current of up to 3.3×10⁻³ amperes in1 cm³ (i.e., 1 mL) can be generated. Therefore, the maximum amount ofenzyme used in a sensor that limits the amount of electrolysis bycontrolling the amount of enzyme should be 1 unit/cm³ or less.

The rate of electrolysis may also be limited by using a relatively smallworking electrode area. When the working electrode area is sufficientlysmall (e.g., no more than about 0.01 cm², no more than about 0.0025 cm²,or no more than about 0.001 cm²), then radial diffusion of analyte tothe electrode may result in a steady-state current, at a constantapplied potential, that is representative of the analyte concentration.For circular electrodes, the appropriate surface area may be achievedusing an electrode with a radius of no more than 60 μm, no more than 30μm, or no more than 20 μm. Radial diffusion of the analyte includestransport of analyte from all directions and not just from the directionnormal to the electrode surface and can, therefore, reduce or preventdepletion of analyte near the electrode surface. A small electrode on aplanar surface permits radial diffusion. In a sensor having a largersurface area electrodes, the transport of analyte to the electrode maybe modeled as semi-infinite linear diffusion instead of radialdiffusion. Thus, the transport of the analyte to the electrode isdominated by diffusion from the direction normal to the electrodesurface. As a result, the reduced transport rate is typically unable toovercome the depletion of analyte near the electrode surface, and atconstant applied potential the current decreases with time, t, accordingto t^(−1/2).

For a potentiometric assay of the type proposed by Yarnitzky and Heller,J. Phys. Chem., 102:10057-61 (1998), in which the potential varieslinearly with the analyte concentration, the concentration of theanalyte and/or redox mediator in a particular oxidation state shouldvary no more than about 20% during the assay. If the concentrationvaries by more than 20%, then the diffusion of the analyte or redoxmediator should be controlled by, for example, controlling temperatureand/or volume of the sample chamber and/or measurement zone.

While this description has described electrolysis of an analyte, oneskilled in the art would recognize that the same devices and techniqueswould also be suitable for measurements of the average oxidation stateof the mediator, such as, for example, in Cottrell types of reactions.

Air-Oxidizable Redox Mediators

In a sensor having a redox mediator, a potential source of measurementerror is the presence of redox mediator in an unknown mixed oxidationstate (i.e., mediator not reproducibly in a known oxidation state). Thecharge passed when the redox mediator is electrooxidized orelectroreduced at the working electrode is affected by its initialoxidation state. Referring to equations (1) and (2) discussed aboveunder the section entitled “Operation of the Sensor,” the current notattributable to the oxidation of biochemical B will flow because ofelectrooxidation of that portion of the redox mediator, A, that is inits reduced form prior to the addition of the sample. Thus, it may beimportant to know the oxidation state of the analyte prior tointroduction of the sample into the sensor. Furthermore, it is desirablethat all or nearly all of the redox mediator have the same state orextent of oxidation prior to the introduction of the sample into thesensor.

Each redox mediator has a reduced form or state and an oxidized form orstate. It is preferred that the amount of redox mediator in the reducedform prior to the introduction of sample be significantly smaller thanthe expected amount of analyte in a sample in order to avoid asignificant background contribution to the measured current. In thisembodiment of the invention, the molar amount of redox mediator in thereduced form prior to the introduction of the analyte is preferably nomore than, on a stoichiometric basis, about 10%, and more preferably nomore than about 5%, and most preferably no more than 1%, of the molaramount of analyte for expected analyte concentrations. (The relativemolar amounts of analyte and redox mediator are compared based on thestoichiometry of the applicable redox reaction. If, for example, twomoles of redox mediator are needed to electrolyze one mole of analyte,then the molar amount of redox mediator in the reduced form prior tointroduction of the analyte is preferably no more than 20% and morepreferably no more than about 10% and most preferably no more than about2% of the molar amount of analyte for expected analyte concentrations.)Methods for controlling the amount of reduced mediator are discussedbelow.

In another aspect of the invention, it is preferred that the ratio ofthe amounts of oxidized redox mediator to reduced redox mediator, priorto introduction of the sample in the sensor, be relatively constantbetween similarly constructed sensors. Any deviation from holding theratio relatively constant may increase the scatter of the resultsobtained for the same sample with multiple similarly made sensors. Forthis aspect of the invention, the percentage of the redox mediator inthe reduced form prior to introduction of the sample in the sensorvaries by no more than about 20% and preferably no more than about 10%between similarly constructed sensors.

One method of controlling the amount of reduced redox mediator prior tothe introduction of the sample in the sensor is to provide an oxidizerto oxidize the reduced form of the mediator. One of the most convenientoxidizers is O₂. Oxygen is usually readily available to perform thisoxidizing function. Oxygen can be supplied by exposing the sensor toair. In addition, most polymers and fluids absorb O₂ from the air unlessspecial precautions are taken. Typically, at least 90% of anair-oxidizable (i.e., O₂ oxidizable) mediator in the solid state is inthe oxidized state upon storage or exposure to air for a useful periodof time, e.g., one month or less, and preferably, one week or less, and,more preferably, one day or less. The air oxidation may take place ineither the solid state or as a solution stored for a time periodsufficient to air oxidize the mediator before deposition on the sensor.In the case of air oxidizable redox mediators in solution, it isdesirable that the time required to achieve at least 80%, preferably atleast 90%, oxidation of the redox mediator is at least 10 times theexpected duration of the assay and is also less than the pot life of thesolution. Preferably, at least 80%, more preferably at least 90%, of theredox mediator is air oxidized in less than 1 week, preferably, in lessthan 1 day, more preferably, in less than 8 hours, and even morepreferably, in less than 1 hour.

While it is desirable to bring the mediators of the sensors manufacturedin a single batch to the same state or extent of oxidation, it is notnecessary that the mediator be completely oxidized to the higher-valentstate. Additionally, it is desirable that the air oxidation of thedissolved redox mediator should not be so fast that air-oxidation duringthe assay can interfere with or introduce error into the measurements.

Suitable mediators which are both air-oxidizable (i.e., O₂-oxidizable)and have electron transfer capabilities have been described hereinabove.One particular family of useful mediators are osmium complexes which arebound to electron-rich nitrogen-containing heterocycles or a combinationof electron-rich nitrogen-containing heterocycles and halides.Electron-rich nitrogen-containing heterocycles include, but are notlimited to, imidazole derivatives and pyridine or phenanthrolinederivatives that contain electron-donating substituents such as alkyl,alkoxy, amino, alkylamino, amido and mercapto groups. Preferably, theosmium complexes have no more than one halide coordinated to the metal,so that the mediators are overall positively charged and thus is arewater soluble. An example is osmium complexed with mono-, di-, andpolyalkoxy-2,2′-bipyridine. Other examples include mono-, di-, andpolyalkoxy-1,10-phenanthroline, where the alkoxy groups have a carbon tooxygen ratio sufficient to retain solubility in water, areair-oxidizable. These osmium complexes typically have two substitutedbipyridine or substituted phenanthroline ligands, the two ligands notnecessarily being identical. These osmium complexes are furthercomplexed with a monomeric or polymeric ligand with one or morenitrogen-containing heterocycles, such as pyridine and imidazole.Preferred polymeric ligands include poly(4-vinyl pyridine) and, morepreferably, poly(1-vinyl imidazole) or copolymers thereof.[Os[4,4′-dimethoxy-2,2′-bipyridine]₂Cl]^(+/+2) complexed with apoly(1-vinyl imidazole) or poly(4-vinyl pyridine) has been shown to beparticularly useful as the Os⁺² cation is oxidizable by O₂ to Os⁺³.Similar results are expected for complexes of[Os(4,7-dimethoxy-1,10-phenanthroline)₂Cl]^(+/+2), and other mono-, di-,and polyalkoxy bipyridines and phenanthrolines, with the same polymers.Other halogen groups such as bromine may be substituted for chlorine.Similar results are also expected for complexes comprising the followingstructures, as specified above:

A complication associated with air-oxidizable mediators arises if theair oxidation of the redox mediator is so fast that a substantialportion of the analyte-reduced redox mediator is oxidized by O₂ duringan analyte assay. This will result in an inaccurate assay as the amountof analyte will be underestimated because the mediator will be oxidizedby air rather than by its electrooxidation at the electrode. It ispreferred that the reaction of the redox mediator with O₂ proceeds moreslowly than the electrooxidation of the mediator, because if the airoxidation of the mediator were fast, then dissolved air and thein-diffusion of air might affect the outcome of the measurement.

Because typically the assay takes about 10 minutes or less, preferably 5minutes or less, and most preferably about 1 minute or less, it ispreferred that the mediator, though air oxidizable in storage, will notbe oxidized by dissolved oxygen during the time of the assay. Thus,mediators that are not air oxidized in 1 minute, and preferably not evenin 10 minutes when dissolved in plasma or in serum, are preferred.Typically, less than 5%, and preferably less than 1%, of the reducedmediator should be oxidized by air during an assay.

The reaction rate of the air oxidation of the mediator can be controlledthrough choice of an appropriate complexing polymer. For example, theoxidation reaction is much faster for[Os(4,4′-dimethoxy-2,2′-bipyridine)₂Cl]^(+/+2) coordinatively coupled topoly(1-vinyl imidazole) than for the same Os complex coupled topoly(4-vinyl pyridine). The choice of an appropriate polymer will dependon the expected analyte concentration and the potential applied betweenthe electrodes, both of which determine the rate of the electrochemicalreaction.

Thus, in one embodiment of the invention, the preferred redox mediatorhas the following characteristics: 1) the mediator does not react withany molecules in the sample or in the sensor other than the analyte(optionally, via a second electron transfer agent); 2) nearly all of theredox mediator is oxidized by an oxidizer such as O₂ prior tointroduction of the sample in the sensor; and 3) the oxidation of theredox mediator by the oxidizer is slow compared to the electrooxidationof the mediator by the electrode.

Alternatively, if the redox mediator is to be oxidized in the presenceof the analyte and electroreduced at the electrode, a reducer ratherthan an oxidizer would be required. The same considerations for theappropriate choice of reducer and mediator apply as describedhereinabove for the oxidizer.

The use of stable air-oxidizable redox mediators in the electrochemicalsensors of the invention provides an additional advantage during storageand packaging. Sensors of the invention which include air-oxidizableredox mediators can be packaged in an atmosphere containing molecularoxygen and stored for long periods of time, e.g., greater than onemonth, while maintaining at least 80% and preferably at least 90% of theredox species in the oxidized state.

Use of the Air-Oxidizable Mediators in Optical Sensors

The air-oxidizable redox species of the present invention can be used inother types of sensors. The osmium complexes described hereinabove aresuitable for use in optical sensors, due to the difference in theabsorption spectra, luminescence and/or fluorescence characteristics ofthe complexed Os⁺² and Os⁺³ species. Absorption, transmission,reflection, luminescence and/or fluorescence measurements of the redoxspecies will correlate with the amount of analyte in the sample (afterreaction between an analyte and the redox species, either directly, orvia a second electron transfer agent such as an enzyme). In thisconfiguration, the molar amount of redox mediator should be greater, ona stoichiometric basis, than the molar amount of analyte reasonablyexpected to fill the measurement zone of the sensor.

Standard optical sensors, including light-guiding optical fiber sensors,and measurement techniques can be adapted for use with theair-oxidizable mediators. For example, the optical sensors of theinvention may include a light-transmitting or light reflecting supporton which the air-oxidizable redox species, and preferably ananalyte-responsive enzyme, is coated to form a film. The support filmforms one boundary for the measurement zone in which the sample isplaced. The other boundaries of the measurement zone are determined bythe configuration of the cell. Upon filling the measurement zone with ananalyte-containing sample, reduction of the air-oxidizable mediator bythe analyte, preferably via reaction with the analyte-responsive enzyme,causes a shift in the mediator's oxidation state that is detected by achange in the light transmission, absorption, or reflection spectra orin the luminescence and/or fluorescence of the mediator at one or morewavelengths of light.

Multiple Electrode Sensors and Calibration

Multiple electrode sensors may be used for a variety of reasons. Forexample, multiple electrode sensors may be used to test a variety ofanalytes using a single sample. One embodiment of a multiple electrodesensor, shown in FIG. 5, has one or more sample chambers which in turnmay contain one or more working electrodes 22 with each workingelectrode 22 defining a different measurement zone. If the redoxmediator is non-leachable, one or more of the working electrodes havethe appropriate chemical reagents, for example, an appropriate enzyme,to test a first analyte and one or more of the remaining workingelectrodes have appropriate chemical reagents to test a second analyte.The chemical reagents (e.g., redox mediator and/or second electrontransfer agent) can be deposited as a sensing layer on the workingelectrode or, if diffusible reagents are used, they can be deposited onany surface of the sample chamber or placed in the sample. For example,a multiple electrode sensor might include 1) one or more workingelectrodes having glucose oxidase in the sensing layer to determineglucose concentration and 2) one or more working electrodes havinglactate oxidase in the sensing layer to determine lactate concentration.

Multiple electrode sensors may also be used to improve the precision ofthe resulting readings. The measurements from each of the workingelectrodes (all or which are detecting the same analyte) can be averagedtogether to obtain a more precise reading. In some cases, measurementsmay be rejected if the difference between the value and the averageexceeds a threshold limit. This threshold limit may be, for example,determined based on a statistical parameter, such as the standarddeviation of the averaged measurements. The average may then berecalculated while omitting the rejected values. Furthermore, subsequentreadings from an electrode that produced a rejected value may be ignoredin later tests if it is assumed that the particular electrode is faulty.Alternatively, a particular electrode may be rejected only after havinga predetermined number of readings rejected based on the readings fromthe other electrodes.

In addition to using multiple electrode sensors to increase precision,multiple measurements may be made at each electrode and averagedtogether to increase precision. This technique may also be used with asingle electrode sensor to increase precision.

Errors in assays may occur when mass produced sensors are used becauseof variations in the volume of the measurement zone of the sensors. Twoof the three dimensions of the measurement zone, the length and thewidth, are usually relatively large, between about 1-5 mm. Electrodes ofsuch dimensions can be readily produced with a variance of 2% or less.The submicroliter measurement zone volume requires, however, that thethird dimension be smaller than the length or width by one or two orderof magnitude. As mentioned hereinabove, the thickness of the samplechamber is typically between about 50 and about 200 μm. Manufacturingvariances in the thickness may be on the order of 20 to 50 μm.Therefore, it may be desirable that a method be provided to accommodatefor this uncertainty in the volume of sample within the measurementzone.

In one embodiment of the invention, depicted in FIG. 5, multiple workingelectrodes 42, 44, 46 are provided on a base material 48. Theseelectrodes are covered by another base, not shown, which has counterelectrodes, not shown, disposed upon it to provide multiple facingelectrode pairs. The variance in the separation distance between theworking electrode and the counter electrode among the electrode pairs ona given sensor is significantly reduced, because the working electrodesand counter electrodes are each provided on a single base with the samespacer 28 between each electrode pair (see FIG. 3).

One example of a multiple electrode sensor that can be used toaccurately determine the volume of the measurement zones of theelectrode pairs and that is also useful in reducing noise is presentedherein. In this example, one of the working electrodes 42 is preparedwith a non-leachable redox mediator and a non-leachable second electrontransfer agent (e.g., an enzyme). Sorbent material may be disposedbetween that working electrode 42 and its corresponding counterelectrode. Another working electrode 44 includes non-leachable redoxmediator, but no second electron transfer agent on the electrode. Again,this second electrode pair may have sorbent material between the workingelectrode 44 and the corresponding counter electrode. An optional thirdworking electrode 46 has no redox mediator and no second electrontransfer agent bound to the electrode, nor is there sorbent materialbetween the working electrode 46 and its corresponding counterelectrode. A similar configuration can be constructed using diffusibleredox mediator and/or diffusible second electron transfer agent althoughdiffusible components are not limited to being disposed on the workingelectrode. In some instances, the distance between electrode pairs issufficient that redox mediator and/or enzyme do not substantiallydiffuse between electrode pairs within the measurement period and/or inthe time period from introduction of the same sample into the samplechamber to the end of the measurement.

The sensor error caused by redox mediator in a non-uniform oxidationstate prior to the introduction of the sample can be measured byconcurrently electrolyzing the sample in the measurement zones that areproximate electrodes 42 and 44. At electrode 42, the analyte iselectrolyzed to provide the sample signal. At electrode 44, the analyteis not electrolyzed because of the absence of the second electrontransfer agent (assuming that a second electron transfer agent isnecessary). However, a charge will pass (and a current will flow) due tothe electrolysis of the redox mediator that was in a mixed oxidationstate (i.e., some redox centers in the reduced state and some in theoxidized state) prior to the introduction of the sample and/or theshuttling of a diffusible redox mediator between the working electrodeand the counter electrode. The small charge passed between theelectrodes in this second electrode pair can be subtracted from thecharge passed between the first electrode pair to substantially removethe error due to the oxidation state of the redox mediator and/or toremove the background current caused by a diffusible redox mediator.This procedure also reduces the error associated with other electrolyzedinterferents, such as ascorbate, urate, and acetaminophen, as well aserrors associated with capacitive charging and faradaic currents.

The thickness of the sample chamber can be determined by measuring thecapacitance, preferably in the absence of any fluid, between electrode46 (or any of the other electrodes 42, 44 in the absence of sorbentmaterial) and its corresponding counter electrode. The capacitance of anelectrode pair depends on the surface area of the electrodes, theinterelectrode spacing, and the dielectric constant of the materialbetween the plates. The dielectric constant of air is unity whichtypically means that the capacitance of this electrode configuration isa few picofarads (or about 100-1000 picofarads if there is fluid betweenthe electrode and counter electrode given that the dielectric constantfor most biological fluids is approximately 75). Thus, since the surfacearea of the electrodes are known, measurement of the capacitance of theelectrode pair allows for the determination of the thickness of themeasurement zone to within about 1-5%.

The amount of void volume in the sorbent material, can be determined bymeasuring the capacitance between electrode 44 (which has no secondelectron transfer agent) and its associated counter electrode, bothbefore and after fluid is added. Upon adding fluid, the capacitanceincreases markedly since the fluid has a much larger dielectricconstant. Measuring the capacitance both with and without fluid allowsthe determination of the spacing between the electrodes and the voidvolume in the sorbent, and thus the volume of the fluid in the reactionzone.

Other electrode configurations can also use these techniques (i.e.,capacitance measurements and coulometric measurements in the absence ofa critical component) to reduce background noise and error due tointerferents and imprecise knowledge of the volume of the interrogatedsample. Protocols involving one or more electrode pairs and one or moreof the measurements described above can be developed and are within thescope of the invention. For example, only one electrode pair is neededfor the capacitance measurements, however, additional electrode pairsmay be used for convenience.

Fill Indicator

When using a sample chamber that is filled with 1 μL or less of fluid,it is often desirable to be able to determine when the sample chamber isfilled. FIGS. 18A-18C illustrate one sensor having a fill indicatorstructure. FIG. 18A illustrates a first substrate 500 upon which aworking electrode 502 is printed. A spacer 504 (FIG. 18B), such as, forexample, a layer of adhesive or a double-sided tape, is formed over thefirst substrate 500 and working electrode 502 with a channel 506 formedin the layer to provide a sample chamber. A second substrate 508 isprinted with two counter electrodes 510, 512, as shown in FIG. 18C(inverted with respect to FIGS. 18A and 18B to show the electrode sideup). In some instances, the counter electrode 510 nearest an entrance514 of the channel 506 has a surface area within the sample chamber thatis at least two times larger than the other counter electrode 512, andpreferably at least five or ten times larger.

The sensor can be indicated as filled by observing a signal between thesecond counter electrode 512 and the working electrode 502 as the sensorfills with fluid. When fluid reaches the second counter electrode 512,the signal from that counter electrode should change. Suitable signalsfor observing include, for example, voltage, current, resistance,impedance, or capacitance between the second counter electrode 512 andthe working electrode 502. Alternatively, the sensor may be observedafter filling to determine if a value of the signal (e.g., voltage,current, resistance, impedance, or capacitance) has been reachedindicating that the sample chamber is filled.

In alternative embodiments, the counter electrode and/or workingelectrode may be divided into two or more parts and the signals from therespective parts observed to determine whether the sensor has beenfilled. In one example, the working electrode is in a facingrelationship with the counter electrode and the indicator electrode. Inanother example, the counter electrode, working electrode, and indicatorelectrode are not in a facing relationship, but may be, for example,side-by-side. In other cases, a second electrode pair may be used withsignals from the second electrode pair being monitored for changesand/or for approaching a particular value to determine that the sensorhas filled. Typically, the indicator electrode is further downstreamfrom a sample inlet port than the working electrode and counterelectrode.

For side-fill sensors, such as those illustrated in FIGS. 19A-19C and20A-20C, two indicator electrodes may be disposed on either side of theprimary counter electrode. This permits the user to fill the samplechamber from either the left or right side with an indicator electrodedisposed further upstream. This three-electrode configuration is notnecessary. Side-fill sensors can also have a single indicator electrodeand, preferably, some indication as to which side should be placed incontact with the sample fluid.

In one embodiment, the use of three counter/reference electrodes and/orindicator electrodes, detects when the sample chamber begins to fill andwhen the sample chamber has been filled to prevent partial filling ofthe sample chamber. In this embodiment, the two indicator electrodes areheld at a different potential than the largest counter/referenceelectrode. The start and completion of filling of the sample chamber isindicated by the flow of current between the indicator andcounter/reference electrodes.

In other instances, the potential of each of the counter/referenceelectrodes may be the same. When the potential at all threecounter/reference electrodes is the same for example, 0 volts, then asthe measurement zone begins to fill, the fluid allows for electricalcontact between a working electrode and the first counter/referenceelectrode, causing a current at the first counter/reference electrodedue to the reaction of the analyte with the enzyme and the mediator.When the fluid reaches the third counter/reference electrode, anothercurrent may be measured similar to the first counter/reference electrodeindicating that the measurement zone is full. When the measurement zoneis full, the three counter/reference electrodes may be shorted togetheror their signals may be added or otherwise combined.

The indicator electrode may also be used to improve the precision of theanalyte measurements according to the methods described above formultiple electrode sensors. The indicator electrode may operate as aworking electrode or as a counter electrode or counter/referenceelectrode. In the embodiment of FIGS. 18A-18C, the indicator electrode512 can act as a second counter or counter/reference electrode withrespect to the working electrode 502. Measurements from the indicatorelectrode/working electrode pair can be combined (for example, added toand/or averaged) with those from the first counter or counter/referenceelectrode/working electrode pair to obtain more accurate measurements.In one embodiment, the indicator electrode may operate as a secondworking electrode with the counter electrode or counter/referenceelectrode. In another embodiment, the indicator electrode may operate asa second working electrode with a second counter electrode orcounter/reference electrode. In still another embodiment, the indicatorelectrode may operate as a second counter electrode or counter/referenceelectrode with a second working electrode.

The sensor or a sensor reader may include a sign (e.g., a visual sign orauditory signal) that is activated in response to the indicatorelectrode to alert the user that the measurement zone has been filled.In some instances, the sensor or a sensor reader may be configured toinitiate a reading when the indicator electrode indicates that themeasurement zone has been filled with or without alerting the user. Thereading can be initiated, for example, by applying a potential betweenthe working electrode and the counter electrode and beginning to monitorthe signals generated at the working electrode.

Heating of Sample

The sample may be heated to increase the rate of diffusion, oxidation,or reduction of the analyte. This heating may be accomplished by avariety of techniques including placing the sensor in a heatedenvironment or applying a heating unit to the sensor.

Another technique includes providing a thermal heating element, such as,for example, a wire or an ink that is capable of converting electricalenergy into heat energy, on the sensor. This wire or ink can be applied,for example, on the opposite side of a base material, such as a polymerfilm, from one or more of the working, counter, reference, orcounter/reference electrodes, or applied around the periphery of theworking, counter, reference, or counter/reference electrodes. In someinstances, the sample may be heated up to 5 to 20° C. above an initialtemperature. In other instances, the temperature of the sample may notbe known but a constant amount of power or current may be applied to thewire or ink.

EXAMPLES

The invention will be further characterized by the following examples.These examples are not meant to limit the scope of the invention whichhas been fully set forth in the foregoing description. Variations withinthe concepts of the invention are apparent to those skilled in the art.

Example 1 Preparation of a Small Volume In Vitro Sensor for theDetermination of Glucose Concentration

A sensor was constructed corresponding to the embodiment of theinvention depicted in FIG. 1. The working electrode was constructed on aMylar™ film (DuPont), the Mylar™ film having a thickness of 0.175 mm anda diameter of about 2.5 cm. An approximately 12 micron thick carbon padhaving a diameter of about 1 cm was screen printed on the Mylar™ film.The carbon electrode was overlaid with a water-insoluble dielectricinsulator (Insulayer) having a thickness of 12 μm, and a 4 mm diameteropening in the center.

The center of the carbon electrode, which was not covered by thedielectric, was coated with a non-leachable redox mediator. The redoxmediator was formed by complexing poly(1-vinyl imidazole) withOs(4,4′-dimethoxy-2,2′-bipyridine)₂Cl₂ followed by cross-linking glucoseoxidase with the osmium polymer using polyethylene glycol diglycidylether (PEGDGE) as described in Taylor et al., J. Electroanal. Chem.,396:511 (1995). The ratio of osmium to imidazole functionalities in theredox mediator was approximately 1:15. The mediator was deposited on theworking electrode in a layer having a thickness of 0.6 μm and a diameterof 4 mm. The coverage of the mediator on the electrode was about 60μg/cm² (dry weight). A spacer material was placed on the electrodesurrounding the mediator-covered surface of the electrode. The spacerwas made of polytetrafluoroethylene (PTFE) and had a thickness of about0.040 mm.

A sorbent material was placed in contact with the mediator-coveredsurface of the working electrode. The sorbent was made of nylon (TetkoNitex nylon 3-10/2). The sorbent had a diameter of 5 mm, a thickness of0.045 mm, and a void volume of about 20%. The volume of sample in themeasurement zone was calculated from the dimensions and characteristicsof the sorbent and the electrode. The measurement zone had a diameter of4 mm (the diameter of the mediator covered surface of the electrode) anda thickness of 0.045 mm (thickness of the nylon sorbent) to give avolume of 0.57 μL. Of this space, about 80% was filled with nylon andthe other 20% was void space within the nylon sorbent. This resultingvolume of sample within the measurement zone was about 0.11 μL.

A counter/reference electrode was placed in contact with the spacer andthe side of the sorbent opposite to the working electrode so that thetwo electrodes were facing each other. The counter/reference electrodewas constructed on a Mylar™ film having a thickness of 0.175 mm and adiameter of about 2.5 cm onto which a 12 micron thick layer ofsilver/silver chloride having a diameter of about 1 cm was screenprinted.

The electrodes, sorbent, and spacer were pressed together using plateson either side of the electrode assembly. The plates were formed ofpolycarbonate plastic and were securely clamped to keep the sensortogether. The electrodes were stored in air for 48 hours prior to use.

Tabs extended from both the working electrode and the counter/referenceelectrode and provided for an electrical contact with the analyzingequipment. A potentiostat was used to apply a potential difference of+200 mV between the working and counter/reference electrodes, with theworking electrode being the anode. There was no current flow between theelectrodes in the absence of sample, which was expected, as noconductive path between the electrodes was present.

The sample was introduced via a small tab of nylon sorbent materialformed as an extension from the nylon sorbent in the sample chamber.Liquid was wicked into the sorbent when contact was made between thesample and the sorbent tab. As the sample chamber filled and the samplemade contact with the electrodes, current flowed between the electrodes.When glucose molecules in the sample came in contact with the glucoseoxidase on the working electrode, the glucose molecules wereelectrooxidized to gluconolactone. The osmium redox centers in the redoxmediator then reoxidized the glucose oxidase. The osmium centers were inturn reoxidized by reaction with the working electrode. This provided acurrent which was measured and simultaneously integrated by a coulometer(EG&G Princeton Applied Research Model #173).

The electrochemical reaction continued until the current reached asteady state value which indicated that greater than 95% of the glucosehad been electroreduced. The current curve obtained by measurement ofthe current at specific intervals was integrated to determine the amountof charge passed during the electrochemical reaction. These charges werethen plotted versus the known glucose concentration to produce acalibration curve.

The sensor was tested using 0.5 μL aliquots of solutions containingknown concentrations of glucose in a buffer of artificial cerebrospinalfluid or in a control serum (Baxter-Dade, Monitrol Level 1, Miami, Fla.)in the range of 3 to 20 mM glucose. The artificial cerebrospinal fluidwas prepared as a mixture of the following salts: 126 mM NaCl, 27.5 mMNaHCO₃, 2.4 mM KCl, 0.5 mM KH₂PO₄, 1.1 mM CaCl₂.2HO, and 0.5 mM Na₂SO₄.

The results of the analyses are shown in Table 1 and in FIG. 7. In Table1, Q_(avg) is the average charge used to electrolyze the glucose in 3-6identical test samples (FIG. 7 graphs the charge for each of the testsamples) and the 90% rise time corresponds to the amount of timerequired for 90% of the glucose to be electrolyzed. The data show asensor precision of 10-20%, indicating adequate sensitivity of thesensor for low glucose concentrations, as well as in the physiologicallyrelevant range (30 μg/dL-600 μg/dL).

TABLE 1 Sensor Results Using Glucose Oxidase Number of Samples 90% risetime Tested Q_(avg) (μC) (sec) buffer only 4  9.9 ± 1.8 13 ± 6  3 mMglucose/buffer 5 17.8 ± 3.5 19 ± 5  6 mM glucose/buffer 4 49.4 ± 4.9 25± 3  10 mM glucose/buffer 6  96.1 ± 12.4 36 ± 17 15 mM glucose/buffer 5205.2 ± 75.7 56 ± 23 20 mM glucose/buffer 4 255.7 ± 41.0 62 ± 17 4.2 mMglucose/serum 3 44.2 ± 4.3 44 ± 3  15.8 mM glucose/serum 3 218.2 ± 57.572 ± 21

The average measured values of glucose concentration were fit by one ormore equations to provide a calibration curve. FIG. 8 shows thecalibration curves for the glucose/buffer data of Table 1. One of the15.0 mM glucose measurements was omitted from these calculations becauseit was more than two standard deviations away from the average of themeasurements. The higher glucose concentrations (10-20 mM) were fit by alinear equation. The lower glucose concentrations were fit by a secondorder polynomial.

FIG. 9 shows the data of Table 1 plotted on an error grid developed byClarke et al., Diabetes Care, 5, 622-27, 1987, for the determination ofthe outcome of errors based on inaccurate glucose concentrationdetermination. The graph plots “true” glucose concentration vs. measuredglucose concentration, where the measured glucose concentration isdetermined by calculating a glucose concentration using the calibrationcurves of FIG. 8 for each data point of FIG. 7. Points in zone A areaccurate, those in zone B are clinically acceptable, and those in zonesC, D, and E lead to increasingly inappropriate and finally dangeroustreatments.

There were 34 data points. Of those data points 91% fell in zone A, 6%in zone B, and 3% in zone C. Only one reading was determined to be inzone C. This reading was off-scale and is not shown in FIG. 9. Thus, 97%of the readings fell in the clinically acceptable zones A and B.

The total number of Os atoms was determined by reducing all of the Osand then electrooxidizing it with a glucose-free buffer in the samplechamber. This resulted in a charge of 59.6±5.4 μC. Comparison of thisresult with the glucose-free buffer result in Table 1 indicated thatless than 20% of the Os is in the reduced form prior to introduction ofthe sample. The variability in the quantity of osmium in the reducedstate is less than 5% of the total quantity of osmium present.

Example 2 Response of the Glucose Sensor to Interferents

A sensor constructed in the same manner as described above for Example 1was used to determine the sensor's response to interferents. The primaryelectrochemical interferents for blood glucose measurements areascorbate, acetaminophen, and urate. The normal physiological ortherapeutic (in the case of acetaminophen) concentration ranges of thesecommon interferents are:

ascorbate: 0.034-0.114 mM

acetaminophen: 0.066-0.200 mM

urate (adult male): 0.27-0.47 mM

Tietz, in: Textbook of Clinical Chemistry, C. A. Burtis and E. R.Ashwood, eds., W.B. Saunders Co., Philadelphia 1994, pp. 2210-12.

Buffered glucose-free interferent solutions were tested withconcentrations of the interferents at the high end of the physiologicalor therapeutic ranges listed above. The injected sample volume in eachcase was 0.5 μL. A potential of +100 mV or +200 mV was applied betweenthe electrodes. The average charge (Q_(avg)) was calculated bysubtracting an average background current obtained from a buffer-only(i.e., interferent-free) solution from an average signal recorded withinterferents present. The resulting average charge was compared with thesignals from Table 1 for 4 mM and 10 mM glucose concentrations todetermine the percent error that would result from the interferent.

TABLE 2 Interferent Response of Glucose Sensors Error @ Error @ 4 mM 10mM Solution E (mV) n Q_(avg) (μC) glucose glucose 0.114 mM ascorbate 1004 0.4 2% <1% 0.114 mM ascorbate 200 4 −0.5 2% <1%  0.2 mM acetaminophen100 4 0.1 <1% <1%  0.2 mM acetaminophen 200 4 1.0 5% 1%  0.47 mM urate100 4 6.0 30% 7%  0.47 mM urate 200 4 18.0 90% 21%

These results indicated that ascorbate and acetaminophen were notsignificant interferents for the glucose sensor configuration,especially for low potential measurements. However, urate providedsignificant interference. This interference can be minimized bycalibrating the sensor response to a urate concentration of 0.37 mM,e.g., by subtracting an appropriate amount of charge as determined byextrapolation from these results from all glucose measurements of thesensor. The resulting error due to a 0.10 mM variation in urateconcentration (the range of urate concentration is 0.27-0.47 in an adultmale) would be about 6% at 4 mM glucose and 100 mV.

Example 3 Sensor with Glucose Dehydrogenase

A sensor similar to that described for Example 1 was prepared and usedfor this example, except that glucose oxidase was replaced bypyrroloquinoline quinone glucose dehydrogenase and a potential of only+100 mV was applied as opposed to the +200 my potential in Example 1.The results are presented in Table 3 below and graphed in FIG. 10.

TABLE 3 Sensor Results Using Glucose Dehydrogenase 90% rise time nQ_(avg) (μC) (s) buffer 4 21.7 ± 5.2 14 ± 3 3 mM glucose/buffer 4  96.9± 15.0 24 ± 6 6 mM glucose/buffer 4 190.6 ± 18.4 26 ± 6 10 mMglucose/buffer 4 327.8 ± 69.3 42 ± 9

The results indicated that the charge obtained from the glucosedehydrogenase sensor was much larger than for the comparable glucoseoxidase sensor, especially for low concentrations of glucose. For 4 mMglucose concentrations the measurements obtained by the two sensorsdiffered by a factor of five. In addition, the glucose dehydrogenasesensor operated at a lower potential, thereby reducing the effects ofinterferent reactions.

In addition, the results from Table 3 were all fit by a linearcalibration curve as opposed to the results in Example 1, as shown inFIG. 10. A single linear calibration curve is greatly preferred tosimplify sensor construction and operation.

Also, assuming that the interferent results from Table 2 are applicablefor this sensor, all of the interferents would introduce an error ofless than 7% for a 3 mM glucose solution at a potential of 100 mV.

Example 4 Determination of Lactate Concentration in a Fluid Stream

The sensor of this Example was constructed using a flow cell(BioAnalytical Systems, Inc. # MF-1025) with a glassy carbon electrode.A redox mediator was coated on the electrode of the flow cell to providea working electrode. In this case, the redox mediator was a polymerformed by complexing poly(1-vinyl imidazole) withOs(4,4′-dimethyl-2,2′-bipyridine)₂Cl₂ with a ratio of 1 osmium for every15 imidazole functionalities. Lactate oxidase was cross-linked with thepolymer via polyethylene glycol diglycidyl ether. The mediator wascoated onto the electrode with a coverage of 500 μg/cm² and a thicknessof 5 μm. The mediator was covered by a polycarbonate track-etchedmembrane (Osmonics-Poretics #10550) to improve adherence in the flowstream. The membrane was then overlaid by a single 50 μm thick spacergasket (BioAnalytical Systems, Inc. #MF-1062) containing a void whichdefined the sample chamber and corresponding measurement zone. Assemblyof the sensor was completed by attachment of a cell block (BioAnalyticalSystems, Inc. #MF-1005) containing the reference and auxiliaryelectrodes of the flow cell.

The sample chamber in this case corresponded to a 50 μm thick cylinder(the thickness of the spacer gasket) in contact with a mediator-coatedelectrode having a surface area of 0.031 cm². The calculated volume ofsample in the measurement zone of this sensor was approximately 0.16 μL.

The flow rate of the fluid stream was 5 μL/min. A standard threeelectrode potentiostat was attached to the cell leads and a potential of+200 mV was applied between the redox mediator-coated glassy carbonelectrode and the reference electrode. This potential was sufficient todrive the enzyme-mediated oxidation of lactate.

As the fluid stream flowed through the sensor, a steady-state currentproportional to the lactate concentration was measured. At periodicintervals the fluid flow was stopped and current was allowed to flowbetween the electrodes until approximately all of the lactate in themeasurement zone was electrooxidized, as indicated by the achievement ofa stabilized, steady-state current. The total charge, Q, required forlactate electrooxidation was found by integration of the differentialcurrent registered from the flow stoppage until the current reached asteady-state. The concentration was then calculated by the followingequation:[lactate]=Q/2FV  (4)where V is the volume of sample within the measurement zone and F isFaraday's constant.

This assay was performed using lactate solutions having nominal lactateconcentrations of TO, 5.0, and 10.0 mM. The measured concentrations forthe assay were 1.9, 5.4, and 8.9 mM respectively.

Example 5 Determination of the Oxidation State ofOs(4,4′-Dimethoxy-2,2′-Bipyridine)₂Cl^(+/+2) Complexed with Poly(1-VinylImidazole)

A sensor having a three electrode design was commercially obtained fromEcossensors Ltd., Long Hanborough, England, under the model name “largearea disposable electrode”. The sensor contained parallel and coplanarworking, reference and counter electrodes. The working surface area (0.2cm²) and counter electrodes were formed of printed carbon and thereference electrode was formed of printed Ag/AgCl. A redox mediator wascoated on the carbon working electrode. The redox mediator was formed bycomplexation of poly(1-vinyl imidazole) withOs(4,4′-dimethoxy-2,2′-bipyridine)₂Cl₂ in a ratio of 15 imidazole groupsper Os cation followed by cross linking the osmium polymer with glucoseoxidase using polyethylene glycol diglycidyl ether.

The electrode was cured at room temperature for 24 hours. The coplanarelectrode array was then immersed in a buffered electrolyte solution,and a potential of +200 mV (sufficient for conversion of Os(II) toOs(III),) was applied between the working electrode and the referenceelectrode.

Upon application of the potential, an undetectable charge of less than 1μC was passed. Subsequent reduction and reoxidation of the redoxmediator yielded a charge for conversion of all Os from Os(II) toOs(III) of 65 μC. Therefore, more than 98% of the Os cations in theredox mediator were in the desired oxidized Os(III) state.

Example 6 Determination of the Oxidation State of theOs(4,4′-Dimethoxy-2,2′-Bipyridine)₂Cl^(+/+2) Complexed with Poly(4-VinylPyridine)

A similar experiment to that of Example 5 was conducted with the sameworking/counter/reference electrode configuration except that the redoxmediator on the working electrode was changed to a complex ofOs(4,4′-dimethoxy-2,2′-bipyridine)₂Cl₂ with poly(4-vinyl pyridine), with12 pyridine groups per Os cation, cross linked with glucose oxidase viapolyethylene glycol diglycidyl ether.

Two sensors were constructed. The electrodes of the two sensors werecured at room temperature for 24 hours. The electrodes were thenimmersed in a buffered electrolyte solution and a potential of +200 mVwas applied between the working and reference electrodes.

Upon application of the potential to the electrodes, a charge of 2.5 μCand 3.8 μC was passed in the two sensors, respectively. Subsequentreduction and reoxidation of the redox mediators yielded oxidationcharges of 27.9 μC and 28.0 μC, respectively. Therefore, the sensorsoriginally contained 91% and 86% of the Os cations in the desirableoxidized Os(III) state.

Example 7 Optical Sensor

An optical sensor is constructed by applying a film of redox polymerwith crosslinked enzyme onto a light-transparent support such as a glassslide. The quantity of redox mediator is equal to or greater than (in astoichiometric sense) the maximum quantity of analyte expected to fillthe measurement zone. The spacer material, sorbent and facing supportare securely clamped. The sample chamber is adapted to transmit lightthrough the assembled sensor to an optical density detector or to aluminescence and/or fluorescence detector. As sample fills the samplechamber and the redox mediator is oxidized, changes in the absorption,transmission, reflection or luminescence and/or fluorescence of theredox mediator in the chamber are correlated to the amount of glucose inthe sample.

Example 8 Blood Volumes from Upper Arm Lancet Sticks

The forearm of a single individual was pierced with a lancet multipletimes in order to determine the reproducibility of blood volumesobtained by this method. Despite more than thirty lancet sticks in theanterior portion of each forearm and the dorsal region of the leftforearm, the individual identified each stick as virtually painless.

The forearm was pierced with a Payless Color Lancet. The blood from eachstick was collected using a 1 μL capillary tube, and the volume wasdetermined by measuring the length of the blood column. The volumesobtained from each stick are shown below in Table 4.

TABLE 4 Volume of Lancet Sticks Left Anterior Right Anterior Left DorsalForearm, (nL) Forearm, (nL) Forearm, (nL) 1 180 190 180 2 250 180 300 3170 120 310 4 150 100 300 5 100 210 60 6 50 140 380 7 90 120 220 8 130140 200 9 120 100 380 10 100 320 11 260 12 250 13 280 14 260 Avg. 138 ±58 nL 140 ± 40 nL 264 ± 83 nL

Example 9 A Sensor with Diffusible Redox Mediator

A sensor was formed by printing graphite ink (Graphite #G4491, Ercon,Wareham, Mass.) on a polyester substrate. A mixture of 5.5 μg/cm²[Os(dimethyoxybipyridine)₂(vinylimidazole)Cl]Cl, 23.7 μg/cm² PQQ-glucosedehydrogenase, and 18.2 μg/cm² Zonyl FSO® surfactant (E. I. duPont deNemours & Co., Inc., Wilmington, Del.) were deposited on a portion ofthe working electrode. A 150 μm thick pressure sensitive adhesive tapewas then applied to the working electrode leaving only a portion of theworking electrode exposed to form a sample chamber. A second polyesterfilm with a counter electrode disposed on the film was provided over thepressure sensitive adhesive tape. The counter electrode was formed bydisposing Ag/AgCl ink (Silver/Silver Chloride #R414, Ercon, Wareham,Mass.) over the second polyester film. The Ag/AgCl counter electrode wascoated with approximately 100 μg/cm² of methylated poly(vinylimidazole)crosslinked using PEGDGE.

Example 10 Measuring Glucose Using Sensor with Diffusible Redox Mediatorat a Potential of 0 V

Sensors were formed as described in Example 9 and used to measureglucose/buffer solutions at 0, 90, 180, 270, and 360 mg/dL glucoseconcentration. The charge measured over time for each of these solutionsis graphed in FIG. 15. In the absence of glucose, the sensor indicatesabout 3 mg/dL glucose concentration. FIG. 16 illustrates the measuredcharge versus glucose concentration for three sensors at each glucoseconcentration. The measured charge varies linearly with glucoseconcentration similar to what is observed for sensors usingnon-leachable redox mediator.

Example 11 Other Sensors Formed Using Diffusible Redox Mediator

Sensors A and B were formed by printing graphite ink (Graphite #G4491,Ercon, Wareham, Mass.) on a polyester substrate. For Sensor A, a mixtureof 8.0 μg/cm² [Os(dimethyoxybipyridine)₂(vinyl imidazole)Cl]Cl, 34.7μg/cm² PQQ-glucose dehydrogenase, and 26.6 μg/cm² Zonyl FSO® surfactant(E.I. duPont de Nemours & Co., Inc., Wilmington, Del.) were deposited ona portion of the working electrode. For Sensor B, a mixture of 24 μg/cm²[Os(dimethyoxybipyridine)₂(vinyl imidazole)Cl]Cl, 104 μg/cm² PQQ-glucosedehydrogenase, and 80 μg/cm² Zonyl FSO® surfactant (E.I. duPont deNemours & Co., Inc., Wilmington, Del.) were deposited on a portion ofthe working electrode. A 200 μm pressure sensitive adhesive tape wasthen formed over the working electrode of each sensor leaving only aportion of the working electrode exposed to form a sample chamber. Asecond polyester film with a counter electrode disposed on the film wasprovided over the pressure sensitive adhesive tape. The counterelectrode of each sensor was formed by disposing Ag/AgCl ink(Silver/Silver Chloride #R414, Ercon, Wareham, Mass.) over the secondpolyester film. The Ag/AgCl counter electrode was coated withapproximately 100 μg/cm² of methylated poly(vinylimidazole) crosslinkedusing PEGDGE.

Example 12 Varying the Amount of Diffusible Redox Mediator in the Sensor

Sensors A and B were each tested to determine the amount of timerequired for electrolysis of the analyte. FIG. 17 illustrates theresults. Increasing the amount of diffusible redox mediator in thesample decreases the response time of the sensor.

Example 13 Clinical Accuracy of the Small Volume Sensor

The sensor of this Example was constructed corresponding to theembodiment of the invention depicted in FIGS. 24A, 24B, and 24C. Thecarbon working electrode was printed on a Melinex™ polyester film(DuPont, Wilmington, Del.), as described in Example 11. The carbonelectrode was coated with 18 μg/cm² Os[(MeO)₂bpy]₂(1-vinylimidazole)Cl₃, 162 μg/cm² GDH (Toyobo, Japan), 1.35 μg/cm² PQQ (Fluka,Mila, Wis.), and 60 μg/cm² Zonyl FSO (DuPont, Wilmington, Del.). Thecoatings were applied to the working electrode at 18° C. and in 50%relative humidity. An adhesive (50 μm thickness) was placed on thecarbon electrode surrounding the coated surface and forming a channelhaving a width of about 0.04 inches.

Two Ag/AgCl counter/reference electrodes were printed on a secondMelinex™ polymer film, as described in Example 11. The film was thenbrought into contact with the adhesive and the working electrode film sothat the working electrode and two counter electrodes were facing eachother. The counter/reference electrodes were coated with 142 μg/cm²methylated polyvinyl imidazole, 18 μg/cm² PEGDGE (PolySciences,Warington, Pa.), and 7 μg/cm² Zonyl FSO (DuPont, Wilmington, Del.). Oneof the counter electrodes, upstream of the other counter electrode, wasused as an indicator electrode to determine when the sample chamber wasfull. The sensors were laminated by three passes with a hand roller andaged for three days at room temperature over CaSO₄.

The sensors were constructed so that when sufficient current flowedbetween indicator and counter/reference electrodes, an external circuitemitted a visual signal indicating that the channel overlying theworking electrode was full of blood.

A few days prior to using the sensors, dry capacitance measurements weretaken to determine the uniformity of the sample chamber volume. Thevariation in capacitance reflected misalignment of electrodes and/orvariation in adhesive thickness. The mean capacitance measured was 7.49pF with a standard deviation of 0.28 pF or 3.8%. The maximum capacitancemeasured was 8.15 pF and the minimum capacitance measured was 6.66 pF.

The sensors were used to determine the glucose concentration in bloodsamples obtained from 23 people. In the study, the people ranged from 26to 76 years of age, fourteen were men, and nine were women. Six of thepeople were diagnosed with Type 1 diabetes, sixteen were diagnosed withType 2 diabetes, and one person was unknown regarding diabetic status.The people studied had an average hematocrit of 40.7% with a standarddeviation of 3.9%. The maximum hematocrit was 49% and the minimumhematocrit was 33.2%.

One blood sample for each person was collected by pricking the finger ofthe subject. A small volume sensor was filled with this residual blood.

Three blood samples for each person were then collected in small volumesensors by using a 2 mm Carelet™ to lance the arm. If an adequate samplewas not obtained in 10 seconds, the area around the puncture wound waskneaded, and then the sensor was filled. Sixteen of the sixty-ninesamples required that the wound be kneaded.

Three blood samples per person were collected by venipuncture. YSI bloodglucose measurements and hematocrit measurements were taken on at leastone sample. Forty-six small volume sensors were also filled with bloodfrom these samples.

Measurements from the sensor were performed at an applied potential of 0mV. BAS potentiostats (CV50W, West Lafayette, Ind.) were “on” before anysample was applied, so that as the strips filled, electrolysis wasimmediate. Current collection was for 150 seconds (this charge is termed“complete” electrolysis), although most assays were essentially completewell before 150 seconds. No results were discarded. Three successivesensor blood glucose measurements were taken.

Measurements for the control samples were performed using YSI bloodglucose measurement (Yellow Springs Instruments, Model 2300 ChemicalGlucose Analyzer).

The data was plotted against YSI venous results and a linear functionwas determined from the data. All data was collected from “complete”(150 second) electrolysis of glucose in the sensor.

FIG. 28 shows the data for 69 small volume sensors tested on bloodobtained from the arm. R² was 0.947, the average CV (coefficient ofvariation) was 4.8%, and the RMS (root mean square) CV was 5.7%.

FIG. 29 shows the data for 23 small volume sensors tested on bloodobtained from the finger. R² was 0.986.

FIG. 30 shows the data for 46 small volume sensors tested on venousblood. R² was 0.986. The average CV was 3.8%. The RMS CV was 4.6%.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it will be apparent toone of ordinarily skill in the art that many variations andmodifications may be made while remaining within the spirit and scope ofthe invention.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyincorporated by reference.

1. A coulometric sensor for determining the concentration of glucose ina sample fluid, the sensor comprising: a working electrode and a counterelectrode, wherein the working electrode and counter electrode areseparated by an effective distance in a range of 25 to 1000 μm; a samplechamber for holding the sample fluid, the sample chamber comprising theworking electrode, the counter electrode, a first indicator electrode toindicate when the sample chamber is beginning to fill with sample, and asecond indicator electrode to indicate when the sample chamber issubstantially filled with sample, wherein the sample chamber is sized tocontain a volume of no more than about 1 μL of the sample; and aglucose-responsive enzyme and a diffusible redox mediator disposed inthe sample chamber, wherein a background signal generated by thediffusible redox mediator in the absence of glucose is no more than 25%of the signal generated by oxidation or reduction of 80 mg/dL ofglucose.
 2. The sensor of claim 1, wherein a background signal generatedby the diffusible redox mediator in the absence of glucose is no morethan 5% of the signal generated by oxidation or reduction of 80 mg/dL ofglucose.
 3. The sensor of claim 1, wherein the sensor comprises: (a) afirst substrate having a proximal end and a distal end, the firstsubstrate defining a first side edge and a second side edge of theelectrochemical sensor extending from the proximal end to the distal endof the first substrate, the distal end being configured and arranged forinsertion into a sensor reader; (b) a second substrate disposed over thefirst substrate, the working electrode being disposed on one of thefirst and second substrates and the counter electrode being disposed onone of the first and second substrates; (c) a spacer disposed betweenthe first and second substrates and defining a first aperture along thefirst side edge of the sensor and a second aperture along the secondside edge of the sensor, the sample chamber extending from the firstaperture to the second aperture; and (d) the first and second indicatorelectrodes disposed on at least one of the first and second substratesand positioned relative to the sample chamber to determine when thesample chamber is beginning to fill with sample and when the samplechamber is substantially filled with sample.
 4. The sensor of claim 1,wherein the sensor comprises: (a) a first substrate having a proximalend and a distal end, the distal end being configured and arranged forinsertion into a sensor reader, the first substrate defining a firstside edge and a second side edge of the electrochemical sensor extendingfrom the proximal end to the distal end of the first substrate; (b) asecond substrate disposed over the first substrate, the workingelectrode being disposed on one of the first and second substrates andthe counter electrode being disposed on one of the first and secondsubstrates; (c) a spacer disposed between the first and secondsubstrates and defining a first aperture along the proximal end of thesensor and a second aperture along the first side edge of the sensor,the sample chamber extending from the first aperture to the secondaperture; and (d) the first and second indicator electrodes disposed onat least one of the first and second substrates and positioned relativeto the sample chamber to determine when the sample chamber is beginningto fill with sample and when the sample chamber is substantially filledwith sample.
 5. The sensor of claim 1, wherein one of the first orsecond indicator electrodes is also a working electrode or a counterelectrode.
 6. The sensor of claim 1, further comprising a visual orauditory sign, coupled to the second indicator electrode, that activateswhen the indicator electrode indicates that the sample chamber containssample.
 7. The sensor of claim 1, wherein the indicator electrodes aredisposed in facing relationship to one of the working electrode and thecounter electrode.
 8. The sensor of claim 1, wherein either the workingelectrode or the counter electrode is disposed between the two indicatorelectrodes.
 9. The sensor of claim 1, wherein at least a portion of theworking electrode is within an effective distance of no more than 200 μmof a portion of the counter electrode.
 10. The sensor of claim 1,wherein the glucose-responsive enzyme is a glucose dehydrogenase. 11.The sensor of claim 1, wherein, within the sensor, the effectivediffusion coefficient of the redox mediator through the sample fluid isless than the effective diffusion coefficient of the glucose through thesample fluid and, preferably, at least ten times less than the effectivediffusion coefficient of the glucose through the sample fluid.
 12. Thesensor of claim 1, wherein the diffusible mediator has a molecularweight of at least 5,000 daltons.
 13. The sensor of claim 1, wherein thesensor is configured and arranged so that the redox mediator is morereadily electrolyzed on the working electrode than the counterelectrode.
 14. The sensor of claim 1, wherein the sensor comprises amolar amount of the redox mediator that is, on a stoichiometric basis,no more than an average normal physiological amount of the glucose and,preferably, the sensor comprises a molar amount of the redox mediatorthat is, on a stoichiometric basis, no more than 20% of an averagenormal physiological amount of the glucose.
 15. The sensor of claim 1,wherein the working electrode has a surface area of no more than about0.01 cm² exposed in the sample chamber.
 16. The sensor of claim 1,wherein the activity of the enzyme is no more than 1 unit/cm³.
 17. Thesensor of claim 1, wherein the sensor is configured and arranged so thatthe diffusible redox mediator precipitates when reacted at the counterelectrode.
 18. The sensor of claim 1, wherein the sensor is configuredand arranged so that a mathematical product of the effective diffusioncoefficient of the redox mediator and the concentration of the redoxmediator is no more than 1×10⁻¹² moles cm⁻¹ sec⁻¹ when sample fluidfills the sample chamber.
 19. The sensor of claim 1, wherein thediffusible redox mediator is disposed on the working electrode.
 20. Thesensor of claim 1, wherein the glucose-responsive enzyme is disposed onthe working electrode.
 21. A method for determining a concentration ofglucose in a sample, comprising the steps of: contacting a sample with asensor, wherein the sensor comprises: a working electrode and a counterelectrode, wherein the working electrode and counter electrode areseparated by an effective distance in a range of 25 to 1000 μm; a samplechamber for holding the sample fluid, the sample chamber comprising theworking electrode, the counter electrode, a first indicator electrode toindicate when the sample chamber is beginning to fill with sample, and asecond indicator electrode to indicate when the sample chamber issubstantially filled with sample, wherein the sample chamber is sized tocontain a volume of no more than about 1 μL of the sample; and aglucose-responsive enzyme and a diffusible redox mediator disposed inthe sample chamber, wherein a background signal generated by thediffusible redox mediator in the absence of glucose is no more than 25%of the signal generated by oxidation or reduction of 80 mg/dL ofglucose; generating a sensor signal at the working electrode, anddetermining the concentration of the glucose by coulometry using thesensor signal.
 22. The method according to claim 21, wherein thebackground signal that is generated by the diffusible redox mediator inthe absence of glucose is no more than 5% of the signal generated byoxidation or reduction of 80 mg/dL of glucose.
 23. The method accordingto claim 21, further comprising: providing calibration data on a batchof the electrochemical sensors to a measurement instrument, saidcalibration data comprising information related to a magnitude of abackground charge for the batch of the electrochemical sensors; whereinthe step of determining the concentration of the glucose comprisesdetermining the concentration of the glucose using the sensor signal andthe calibration data.
 24. A method for determining a concentration ofglucose in a sample, the method comprising the steps of: contacting asample with a sensor, wherein the sensor comprises: a working electrodeand a counter electrode, wherein the working electrode and counterelectrode are separated by an effective distance in a range of 25 to1000 μm; a sample chamber for holding the sample fluid, the samplechamber comprising the working electrode, the counter electrode, a firstindicator electrode to indicate when the sample chamber is beginning tofill with sample, and a second indicator electrode to indicate when thesample chamber is substantially filled with sample, wherein the samplechamber is sized to contain a volume of no more than about 1 μL of thesample; and a glucose-responsive enzyme and a diffusible redox mediatordisposed in the sample chamber, wherein a background signal generated bythe diffusible redox mediator in the absence of glucose is no more than25% of the signal generated by oxidation or reduction of 80 mg/dL ofglucose; observing a signal from the second indicator electrode tosignify that the sample chamber is substantially filled with sample;applying a potential between the working electrode and the counterelectrode to electrolyze the glucose in the sample; generating aglucose-responsive signal from the sensor in response to electrolysis ofthe glucose in the sample; and determining the concentration of theglucose by coulometry using the glucose-responsive signal.